Designed repeat proteins binding to serum albumin

ABSTRACT

New designed repeat proteins with binding specificity for serum albumin are described, as well as nucleic acids encoding such serum albumin binding proteins, pharmaceutical compositions comprising such proteins, the use of such proteins to modify the pharmacokinetics of therapeutic relevant polypeptides and the use of such proteins in the treatment of diseases. The repeat proteins of the invention have a substantially increased half-life in plasma compared to proteins not binding serum albumin.

FIELD OF THE INVENTION

The present invention relates to designed repeat proteins with bindingspecificity for serum albumin, as well as nucleic acids encoding suchserum albumin binding proteins, pharmaceutical compositions comprisingsuch proteins, the use of such proteins to modify the pharmacokineticsof bioactive compounds and the use of such proteins in the treatment ofdiseases.

BACKGROUND OF THE INVENTION

There is a strong interest of the pharmaceutical industry to increasethe effectiveness of bioactive compounds, such as protein therapeutics,by modulating or increasing their pharmacokinetic (PK) in vivoproperties. This is especially true for bioactive compounds that arerapidly eliminated from the circulation by renal clearance. The kidneygenerally filters out molecules from circulation that have an apparentmolecular weight below 60 kDa. One strategy to improve thepharmacokinetic properties of such small bioactive compounds is tosimply increase their apparent molecular size (i.e. to increase theirhydrodynamic radius), e.g. through the addition of non-proteinaceouspolymer moieties such as polyethylene glycol polymers or sugar residuesor the addition of proteinaceous polymer moieties such as globularproteins or unstructured polypeptides, such as those described in WO2007/103515 and WO 2008/155134.

Other strategies harness the long circulation half-life of serumproteins, such as immunoglobulins and serum albumin. Serum albuminhaving a molecular weight of 67 kDa is the most abundant protein inplasma, present at about 50 mg/ml (0.6 mM), and has a serum half-life of19 days in humans. Serum albumin helps to maintain plasma pH,contributes to colloidal blood pressure, functions as carrier of manymetabolites and fatty acids, and serves as a major drug transportprotein in the plasma. There are several major small molecule bindingsites in albumin that have been described.

It has been shown that non-covalent association with serum albumin canextend the half-life of short lived small molecules or polypeptides (WO1991/001743). Polypeptides that are specifically binding to serumalbumin, and that thereby can extend the in vivo half-life of othermolecules coupled to them, include variants of bacterial albumin bindingdomains (e.g. WO 2005/097202 and WO 2009/016043), small peptides (e.g.Dennis, M. S., et al., J. Biol. Chem. 277(3), 35035-43, 2002 and WO2001/045746) and fragments of immunoglobulins (e.g. WO 2008/043822, WO2004/003019; WO 2008/043821; WO 2006/040153; WO 2006/122787 and WO2004/041865). WO 2008/043822 refers to other binding proteins thanfragments of immunoglobulins, such as molecules based on protein Adomains, tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors,designed ankyrin repeats and PDZ domains, which might be generated tospecifically bind to serum albumin. Nevertheless, WO 2008/043822 doesneither disclose the selection of designed ankyrin repeat domains withbinding specificity for serum albumin (SA) nor concrete repeat sequencemotifs of repeat domains that specifically bind to SA. Furthermore, itwas described that the in vivo half-life of polypeptides can beprolonged by their genetic fusion to serum albumin (e.g. WO1991/001743). Such an alteration of the in vivo half-life of drugs maypositively alter their pharmacokinetic (PK) and/or pharmacodynamic (PD)properties. This is a key issue in the development of new and efficienttherapeutics and disease treatment methods. There is therefore a need inthe art of new ways of altering PK and/or PD of bioactive compounds.

There are, beside antibodies, novel binding proteins or binding domainsthat can be used to specifically bind a target molecule (e.g. Binz, H.K., Amstutz, P. and Plückthun, A., Nat. Biotechnol. 23, 1257-1268,2005). One such novel class of binding proteins or binding domains arebased on designed repeat proteins or designed repeat domains (WO2002/020565; Binz, H. K., Amstutz, P., Kohl, A., Stumpp, M. T., Briand,C., Forrer, P., Grütter, M. G., and Plückthun, A., Nat. Biotechnol. 22,575-582, 2004; Stumpp, M. T., Binz, H. K and Amstutz, P., Drug Discov.Today 13, 695-701, 2008). WO 2002/020565 describes how large librariesof repeat proteins can be constructed and their general application.Nevertheless, WO 2002/020565 does neither disclose the selection ofrepeat domains with binding specificity for SA nor concrete repeatsequence motifs of repeat domains that specifically bind to SA.Furthermore, WO 2002/020565 does not suggest that repeat domains withbinding specificity for SA could be used to modulate the PK or PD ofother molecules. These designed repeat domains harness the modularnature of repeat proteins and possess N-terminal and C-terminal cappingmodules to prevent the designed repeat domains from aggregation byshielding the hydrophobic core of the domain (Forrer, P., Stumpp, M. T.,Binz, H. K. and Plückthun, A., FEBS letters 539, 2-6, 2003). Thesecapping modules were based on the capping repeats of the naturalguanine-adenine-binding protein (GA-binding protein). It was shown thatthe thermal and thermodynamic stability of these designed ankyrin repeatdomains could be further increased by improving the C-terminal cappingrepeat derived from the GA-binding protein (Interlandi, G., Wetzel, S.K, Settanni, G., Plückthun, A. and Caflisch, A., J. Mol. Biol. 375,837-854, 2008; Kramer, M. A, Wetzel, S. K., Plückthun, A., Mittl, P. R.E, and Grütter, M. G., J. Mol. Biol. 404, 381-391, 2010). The authorsintroduced a total of eight mutations into this capping module andextended its C-terminal helix by adding three distinct amino acids.Nevertheless, the introduction of these modifications in the C-terminalcapping module resulted in a tendency of unwanted dimerization of adesigned repeat domain carrying this mutated C-terminal capping module.Thus, there is a need for the generation of further optimized C-terminalcapping modules or C-terminal capping repeats of ankyrin repeat domains.

Targeting SA to modulate the PK and/or PD with currently availableapproaches is not always effective. It has even become increasinglyapparent that the modulation of the PK and/or PD of molecules byhijacking SA is complex and not yet fully understood.

Overall, a need exists for improved binding proteins with specificityfor SA able to improve the PK and/PD of therapeutic relevant moleculesor polypeptides for treating cancer and other pathological conditions.

The technical problem underlying the present invention is identifyingnovel binding proteins, such as repeat domains with binding specificityto SA, able to modify the PK and/or PD of therapeutic relevant moleculesfor an improved treatment of cancer and other pathological conditions.The solution to this technical problem is achieved by providing theembodiments characterized in the claims.

SUMMARY OF THE INVENTION

The present invention relates to a binding protein comprising at leastone ankyrin repeat domain, wherein said ankyrin repeat domain hasbinding specificity for a mammalian serum albumin and wherein saidankyrin repeat domain comprises an ankyrin repeat module having an aminoacid sequence selected from the group consisting of SEQ ID NO:49, 50, 51and 52 and sequences, wherein up to 9 amino acids in SEQ ID NO:49, 50,51 and 52 are exchanged by any amino acid.

In a further embodiment, the invention relates to a binding proteincomprising at least one ankyrin repeat domain, wherein said repeatdomain has binding specificity for a mammalian serum albumin and whereinsaid ankyrin repeat domain comprises an amino acid sequence that has atleast 70% amino acid sequence identity with one ankyrin repeat domainselected from the group consisting of SEQ ID NOs: 17 to 31 and 43 to 48,wherein G at position 1 and/or S at position 2 of said ankyrin repeatdomain are optionally missing.

In particular, the invention relates to a binding protein as definedherein above, wherein the ankyrin repeat domain competes for binding toa mammalian serum albumin with an ankyrin repeat domain selected fromthe group consisting of SEQ ID NOs: 17 to 31 and 43 to 48.

Furthermore the invention relates to such a binding protein comprising abioactive compound, in particular a binding protein comprising abioactive compound having an at least 2-fold higher terminal plasmahalf-life in a mammal compared to the terminal plasma half-life of saidunmodified bioactive compound.

The invention further relates to nucleic acid molecules encoding thebinding proteins of the present invention, and to a pharmaceuticalcomposition comprising one or more of the above mentioned bindingproteins or nucleic acid molecules.

The invention further relates to a method of treatment of a pathologicalcondition using the binding proteins of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A to 1C. Stability analysis of selected DARPins by SEC.

Elution profiles of size exclusion chromatography (SEC) runs of DARPinswith specificity for xSA before (FIG. 1A), after incubation at 30 mg/ml(˜2 mM) in PBS for 28 days at 40° C. (FIG. 1B) or after storage for 1month at −80° C. (FIG. 1C) analyzed with a Superdex 200 column 5/150(FIG. 1A or FIG. 1B) or with a superdex200 10/300GL (FIG. 1C). Allsamples were expressed and purified as described in Example 1. For SECanalysis samples were diluted to a concentration of 500 μM. Themolecular mass standards Aprotinin (AP) 6.5 kDa, Carbonic Anhydrase (CA)29 kDa and Conalbumin (CO) 75 kDa are indicated by arrows.

xSA, mammalian serum albumin, A, absorbance at 280 nm; t, retention timein minutes; DARPin #19 (SEQ ID NO:19 with a His-tag (SEQ ID NO:15) fusedto its N-terminus); DARPin #20 (SEQ ID NO:20 with a His-tag (SEQ IDNO:15) fused to its N-terminus); DARPin #21 (SEQ ID NO:21 with a His-tag(SEQ ID NO:15) fused to its N-terminus); DARPin #22 (SEQ ID NO:22 with aHis-tag (SEQ ID NO:15) fused to its N-terminus); DARPin #27 (SEQ IDNO:27 with a His-tag (SEQ ID NO:15) fused to its N-terminus); DARPin #28(SEQ ID NO:28 with a His-tag (SEQ ID NO:15) fused to its N-terminus);DARPin #29 (SEQ ID NO:29 with a His-tag (SEQ ID NO:15) fused to itsN-terminus); DARPin #30 (SEQ ID NO:30 with a His-tag (SEQ ID NO:15)fused to its N-terminus). DARPin #43 (SEQ ID NO:43 with a His-tag (SEQID NO:15) fused to its N-terminus). DARPin #44 (SEQ ID NO:44 with aHis-tag (SEQ ID NO:15) fused to its N-terminus). DARPin #45 (SEQ IDNO:45 with a His-tag (SEQ ID NO:15) fused to its N-terminus). DARPin #46(SEQ ID NO:46 with a His-tag (SEQ ID NO:15) fused to its N-terminus).DARPin #47 (SEQ ID NO:47 with a His-tag (SEQ ID NO:15) fused to itsN-terminus). DARPin #48 (SEQ ID NO:48 with a His-tag (SEQ ID NO:15)fused to its N-terminus).

FIGS. 2A to 2D. Thermal stability of selected DARPins.

Traces from thermal denaturation of DARPins with specificity for xSA(followed by an increase of the fluorescence intensity of the dye SYPROorange present in the buffer) in PBS at pH 7.4 (FIGS. 2A and 2B) and inMES buffer at pH 5.8 (FIGS. 2C and 2D) (250 mM(2-N-morpholino)ethanesulphonic acid pH 5.5), 150 mM NaCl, mixed withPBS pH 7.4 1 to 4 (v/v) and adjusting the pH to 5.8).

F, relative fluorescence units (RFUs), excitation at 515-535 nm,detection at 560-580 nm; T, temperature in ° C.; Definition of DARPinssee above.

FIGS. 3A and 3B. Plasma clearance of selected DARPins in mice.

The clearance from blood plasma of DARPins with specificity for MSA(mouse serum albumin) and control DARPins were assessed in mice.

(FIG. 3A) DARPins comprising just one repeat domain with bindingspecificity for MSA compared to DARPin #32 (see below) having no bindingspecificity for MSA.

(FIG. 3B) DARPins comprising two protein domains (one of which is arepeat domain with binding specificity for MSA) compared to DARPin #32having no binding specificity for MSA.

DARPins were labeled via the His-Tag with a ^(99m)Tc-carbonyl compoundand injected intravenously in mice. The radioactivity of the blood ofinjected mice was measured at different time points after injection andshown as a ratio of the injected dose corrected for the radioactivedecay of ^(99m)Tc (% ID). The fitted curves show the result ofnon-linear regressions of the radioactivity measured at different timepoints—two phase decay (Graphpad Prism). Each data point indicates theaverage of two mice per group.

% ID, percent injected dose corrected for the radioactive decay of^(99m)Tc; t, time in hours; DARPin #18 (SEQ ID NO:18 with a His-tag (SEQID NO:15) fused to its N-terminus); DARPin #23 (SEQ ID NO:23 with aHis-tag (SEQ ID NO:15) fused to its N-terminus); DARPin #25 (SEQ IDNO:25 with a His-tag (SEQ ID NO:15) fused to its N-terminus); DARPin #32(a negative control DARPin with no binding specificity to xSA, SEQ IDNO:32 with a His-tag (SEQ ID NO:15) fused to its N-terminus);

DARPin #33 (a DARPin comprising two repeat domains, one with bindingspecificity for xSA, SEQ ID NO:33 with a His-tag (SEQ ID NO:15) fused toits N-terminus);

DARPin #35 (a DARPin comprising two repeat domains, one with bindingspecificity for xSA, SEQ ID NO:35 with a His-tag (SEQ ID NO:15) fused toits N-terminus);

DARPin #36 (a DARPin comprising two repeat domains, one with bindingspecificity for xSA, SEQ ID NO:36 with a His-tag (SEQ ID NO:15) fused toits N-terminus).

FIGS. 4A and 4B. Plasma clearance of selected DARPins in cynomolgusmonkeys. The clearance of DARPins with specificity for CSA (cynomolgusmonkey serum albumin) and control DARPins from blood plasma was assessedin cynomolgus monkeys. (FIG. 4A) DARPin #26 was compared to DARPin #32having no binding specificity to CSA. (FIG. 4B) DARPins #24, 34 and 17were compared to DARPin #32 having no binding specificity to CSA. Thefollowing DARPins were intravenously injected in cynomolgus monkeys att=0 hours at a concentration of 0.5 mg/ml (DARPin #26, DARPin #24,DARPin #17 and DARPin #32) or 1 mg/ml (DARPin #34): The concentration ofthe DARPins in the plasma of monkeys was measured by ELISA at differenttime points after injection. The curves show the result of non-linearregressions of the concentrations measured at different time points—twophase decay (Graphpad Prism). From the second phase, the terminal plasmahalf-life of a DARPin can be determined. Each single data pointindicates the average of two independent ELISA measurements of the sameserum sample.

C, DARPin concentration in nM; t, time in hours;

DARPin #17 (SEQ ID NO:17 with a His-tag (SEQ ID NO:15) fused to itsN-terminus); DARPin #24 (SEQ ID NO:24 with a His-tag (SEQ ID NO:15)fused to its N-terminus); DARPin #26 (SEQ ID NO:26 with a His-tag (SEQID NO:15) fused to its N-terminus); DARPin #32 (a negative controlDARPin with no binding specificity to xSA, SEQ ID NO:32 with a His-tag(SEQ ID NO:15) fused to its N-terminus);

DARPin #34 (a DARPin comprising two repeat domains, one with bindingspecificity for xSA, SEQ ID NO:34 with a His-tag (SEQ ID NO:15) fused toits N-terminus).

DETAILED DESCRIPTION OF THE INVENTION

The binding domain according to the invention is specific for amammalian serum albumin (xSA). Preferably, the binding domain accordingto the invention is specific for a serum albumin of mice, rat, dog,rabbit, monkey or human origin. More preferably, the binding domainaccording to the invention is specific for a serum albumin of humanorigin (HSA).

The term “protein” refers to a polypeptide, wherein at least part of thepolypeptide has, or is able to acquire a defined three-dimensionalarrangement by forming secondary, tertiary, or quaternary structureswithin and/or between its polypeptide chain(s). If a protein comprisestwo or more polypeptides, the individual polypeptide chains may belinked non-covalently or covalently, e.g. by a disulfide bond betweentwo polypeptides. A part of a protein, which individually has, or isable to acquire, a defined three-dimensional arrangement by formingsecondary or tertiary structures, is termed “protein domain”. Suchprotein domains are well known to the practitioner skilled in the art.

The term “recombinant” as used in recombinant protein, recombinantprotein domain, recombinant binding protein and the like, means thatsaid polypeptides are produced by the use of recombinant DNAtechnologies well known by the practitioner skilled in the relevant art.For example, a recombinant DNA molecule (e.g. produced by genesynthesis) encoding a polypeptide can be cloned into a bacterialexpression plasmid (e.g. pQE30, Qiagen), yeast expression plasmid ormammalian expression plasmid. When, for example, such a constructedrecombinant bacterial expression plasmid is inserted into an appropriatebacteria (e.g. Escherichia coli), this bacteria can produce thepolypeptide encoded by this recombinant DNA. The correspondinglyproduced polypeptide is called a recombinant polypeptide.

In the context of the present invention, the term “polypeptide” relatesto a molecule consisting of one or more chains of multiple, i.e. two ormore, amino acids linked via peptide bonds. Preferably, a polypeptideconsists of more than eight amino acids linked via peptide bonds.

The term “polypeptide tag” refers to an amino acid sequence attached toa polypeptide/protein, wherein said amino acid sequence is useful forthe purification, detection, or targeting of said polypeptide/protein,or wherein said amino acid sequence improves the physicochemicalbehavior of the polypeptide/protein, or wherein said amino acid sequencepossesses an effector function. The individual polypeptide tags,moieties and/or domains of a binding protein may be connected to eachother directly or via polypeptide linkers. These polypeptide tags areall well known in the art and are fully available to the person skilledin the art. Examples of polypeptide tags are small polypeptidesequences, for example, His (e.g. the His-tag of SEQ ID NO:15), myc,FLAG, or Strep-tags or moieties such as enzymes (for example enzymeslike alkaline phosphatase), which allow the detection of saidpolypeptide/protein, or moieties which can be used for targeting (suchas immunoglobulins or fragments thereof) and/or as effector molecules.

The term “polypeptide linker” refers to an amino acid sequence, which isable to link, for example, two protein domains, a polypeptide tag and aprotein domain, a protein domain and a non-polypeptide moiety such aspolyethylene glycol or two sequence tags. Such additional domains, tags,non-polypeptide moieties and linkers are known to the person skilled inthe relevant art. A list of example is provided in the description ofthe patent application WO 2002/020565. Particular examples of suchlinkers are glycine-serine-linkers and proline-threonine-linkers ofvariable lengths; preferably, said linkers have a length between 2 and24 amino acids; more preferably, said linkers have a length between 2and 16 amino acids. An example of a glycine-serine-linker is provided inSEQ ID NO:16.

The term “polymer moiety” refers to either a proteinaceous polymermoiety or a non-proteinaceous polymer moiety. A “proteinaceous polymermoiety” preferably is a polypeptide that does not form a stable tertiarystructure while not forming more than 10%, preferably, not more than 5%;also preferred, not more than 2%; even more preferably, not more than1%; and most preferably, no detectable amounts, as determined by sizeexclusion chromatography (SEC) of oligomers or aggregates when stored ata concentration of about 0.1 mM in phosphate buffered saline (PBS) atroom temperature (RT) for one month. Such proteinaceous polymer moietiesrun at an apparent molecular weight in SEC that is higher than theireffective molecular weight when using globular proteins as molecularweight standards for the SEC. Preferably, the apparent molecular weightof said proteinaceous polymer moieties determined by SEC is 1.5×, 2× or2.5× higher than their effective molecular weight calculated from theiramino acid sequence. Also preferably, the apparent molecular weights ofsaid non-proteinaceous polymer moieties determined by SEC is 2×, 4× or8× higher than their effective molecular weight calculated from theirmolecular composition. Preferably, more than 50%, 70% or even 90% of theamino acids of said proteinaceous polymer moiety do not form stablesecondary structures at a concentration of about 0.1 mM in PBS at RT asdetermined by Circular Dichroism (CD) measurements. Most preferably,said proteinaceous polymer shows a typical near UV CD-spectra of arandom coil conformation. Such CD analyses are well known to the personskilled in the art. Also preferable are proteinaceous polymer moietiesthat consist of more than 50, preferably more than 100, 200, 300, 400,500, 600, 700, or most preferably more than 800 amino acids. Examples ofproteinaceous polymer moieties are XTEN® (a registered trademark ofAmunix; WO 2007/103515) polypeptides, or polypeptides comprisingproline, alanine and serine residues as described in WO 2008/155134.Such proteinaceous polymer moieties can be covalently attached to, forexample, a binding domain of the invention by the generation of geneticfusion polypeptides using standard DNA cloning technologies, followed bytheir standard expression and purification.

A polymer moiety of the invention may vary widely in molecular weight(i.e. from about 1 kDa to about 150 kDa). Preferably, the polymer moietyhas a molecular weight of at least 2, more preferably at least 5, 10,20, 30, 50, 70, or most preferably at least 100 kDa. Preferably, saidpolymer moiety is connected by a polypeptide linker to a binding domain.

Examples of non-proteinaceous polymer moieties are hydroxyethyl starch(HES), polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylene. The term “PEGylated” means that a PEG moiety iscovalently attached to, for example, a polypeptide of the invention.

In a specific embodiment, a PEG moiety or any other non-proteinaceouspolymer can, e.g., be coupled to a cysteine thiol via a maleimide linkerwith the cysteine being coupled via a peptide linker to the N- orC-terminus of a binding domain as described herein.

The term “binding protein” refers to a protein comprising one or morebinding domains, one or more bioactive compounds and one or more polymermoieties as further explained below. Preferably, said binding proteincomprises up to four binding domains. More preferably, said bindingprotein comprises up to two binding domains. Most preferably, saidbinding protein comprises only one binding domain. Furthermore, any suchbinding protein may comprise additional protein domains that are notbinding domains, multimerization moieties, polypeptide tags, polypeptidelinkers and/or a single Cys residue. Examples of multimerizationmoieties are immunoglobulin heavy chain constant regions which pair toprovide functional immunoglobulin Fc domains, and leucine zippers orpolypeptides comprising a free thiol which forms an intermoleculardisulfide bond between two such polypeptides. The single Cys residue maybe used for conjugating other moieties to the polypeptide, for example,by using the maleimide chemistry well known to the person skilled in theart. Preferably, said binding protein is a recombinant binding protein.Also preferably, the binding domains of binding protein possessdifferent target specificities.

The term “binding domain” means a protein domain exhibiting the same“fold” (three-dimensional arrangement) as a protein scaffold and havinga predetermined property, as defined below. Such a binding domain may beobtained by rational, or most commonly, combinatorial proteinengineering techniques, skills which are known in the art (Binz et al.,2005, loc. cit.). For example, a binding domain having a predeterminedproperty can be obtained by a method comprising the steps of (a)providing a diverse collection of protein domains exhibiting the samefold as a protein scaffold as defined further below; and (b) screeningsaid diverse collection and/or selecting from said diverse collection toobtain at least one protein domain having said predetermined property.The diverse collection of protein domains may be provided by severalmethods in accordance with the screening and/or selection system beingused, and may comprise the use of methods well known to the personskilled in the art, such as phage display or ribosome display.Preferably, said binding domain is a recombinant binding domain.

The term “protein scaffold” means a protein with exposed surface areasin which amino acid insertions, substitutions or deletions are highlytolerable. Examples of protein scaffolds that can be used to generatebinding domains of the present invention are antibodies or fragmentsthereof such as single-chain Fv or Fab fragments, protein A fromStaphylococcus aureus, the bilin binding protein from Pieris brassicaeor other lipocalins, ankyrin repeat proteins or other repeat proteins,and human fibronectin. Protein scaffolds are known to the person skilledin the art (Binz et al., 2005, loc. cit.; Binz et al., 2004, loc. cit.).

The term “predetermined property” refers to a property such as bindingto a target, blocking of a target, activation of a target-mediatedreaction, enzymatic activity, and related further properties. Dependingon the type of desired property, one of ordinary skill will be able toidentify format and necessary steps for performing screening and/orselection of a binding domain with the desired property. Preferably,said predetermined property is binding to a target.

The definitions hereinafter for repeat proteins are based on those inpatent application WO 2002/020565. Patent application WO 2002/020565further contains a general description of repeat protein features,techniques and applications.

The term “repeat proteins” refers to a protein comprising one or morerepeat domains. Preferably, each of said repeat proteins comprises up tofour repeat domains. More preferably, each of said repeat proteinscomprises up to two repeat domains. Most preferably, each of the repeatproteins comprises only one repeat domain. Furthermore, said repeatprotein may comprise additional non-repeat protein domains, polypeptidetags and/or polypeptide linkers.

The term “repeat domain” refers to a protein domain comprising two ormore consecutive repeat units (modules) as structural units, whereinsaid structural units have the same fold, and stack tightly to create,for example, a superhelical structure having a joint hydrophobic core.Preferably, a repeat domain further comprises an N-terminal and/or aC-terminal capping unit (or module). Even more preferably, saidN-terminal and/or C-terminal capping units (or modules) are cappingrepeats.

The term “designed repeat protein” and “designed repeat domain” refer toa repeat protein or repeat domain, respectively, obtained as the resultof the inventive procedure explained in patent application WO2002/020565. Designed repeat proteins and designed repeat domains aresynthetic and not from nature. They are man-made proteins or domains,respectively, obtained by expression of correspondingly designed nucleicacids. Preferably, the expression is done in eukaryotic or prokaryoticcells, such as bacterial cells, or by using a cell-free in vitroexpression system. Accordingly, a designed ankyrin repeat protein (i.e.a DARPin) corresponds to a binding protein of the invention comprisingat least one ankyrin repeat domain.

The term “structural unit” refers to a locally ordered part of apolypeptide, formed by three-dimensional interactions between two ormore segments of secondary structure that are near one another along thepolypeptide chain. Such a structural unit exhibits a structural motif.The term “structural motif” refers to a three-dimensional arrangement ofsecondary structure elements present in at least one structural unit.Structural motifs are well known to the person skilled in the art.Structural units alone are not able to acquire a definedthree-dimensional arrangement; however, their consecutive arrangement,for example as repeat modules in a repeat domain, leads to a mutualstabilization of neighboring units resulting in a superhelicalstructure.

The term “repeat unit” refers to amino acid sequences comprising repeatsequence motifs of one or more naturally occurring repeat proteins,wherein said “repeat units” are found in multiple copies, and whichexhibit a defined folding topology common to all said motifs determiningthe fold of the protein. Such repeat units correspond to the “repeatingstructural units (repeats)” of repeat proteins as described by Forrer etal., 2003, loc. cit. or the “consecutive homologous structural units(repeats)” of repeat proteins as described by Binz et al, 2004, loc.cit. Such repeat units comprise framework residues and interactionresidues. Examples of such repeat units are armadillo repeat units,leucine-rich repeat units, ankyrin repeat units, tetratricopeptiderepeat units, HEAT repeat units, and leucine-rich variant repeat units.Naturally occurring proteins containing two or more such repeat unitsare referred to as “naturally occurring repeat proteins”. The amino acidsequences of the individual repeat units of a repeat protein may have asignificant number of mutations, substitutions, additions and/ordeletions when compared to each other, while still substantiallyretaining the general pattern, or motif, of the repeat units.

The term “ankyrin repeat unit” shall mean a repeat unit, which is anankyrin repeat as described, for example, by Forrer et al., 2003, loc.cit. Ankyrin repeats are well known to the person skilled in the art.

The term “framework residues” relates to amino acid residues of therepeat units, or the corresponding amino acid residues of the repeatmodules, which contribute to the folding topology, i.e. which contributeto the fold of said repeat unit (or module) or which contribute to theinteraction with a neighboring unit (or module). Such contribution mightbe the interaction with other residues in the repeat unit (or module),or the influence on the polypeptide backbone conformation as found inα-helices or β-sheets, or amino acid stretches forming linearpolypeptides or loops.

The term “target interaction residues” refers to amino acid residues ofthe repeat units, or the corresponding amino acid residues of the repeatmodules, which contribute to the interaction with target substances.Such contribution might be the direct interaction with the targetsubstances, or the influence on other directly interacting residues,e.g. by stabilizing the conformation of the polypeptide of a repeat unit(or module) to allow or enhance the interaction of directly interactingresidues with said target. Such framework and target interactionresidues may be identified by analysis of the structural data obtainedby physicochemical methods, such as X-ray crystallography, NMR and/or CDspectroscopy, or by comparison with known and related structuralinformation well known to practitioners in structural biology and/orbioinformatics.

Preferably, the repeat units used for the deduction of a repeat sequencemotif are homologous repeat units, wherein the repeat units comprise thesame structural motif and wherein more than 70% of the frameworkresidues of said repeat units are homologous to each other. Preferably,more than 80% of the framework residues of said repeat units arehomologous. Most preferably, more than 90% of the framework residues ofsaid repeat units are homologous. Computer programs to determine thepercentage of homology between polypeptides, such as Fasta, Blast orGap, are known to the person skilled in the art. Further preferably, therepeat units used for the deduction of a repeat sequence motif arehomologous repeat units obtained from repeat domains selected on atarget, for example as described in Example 1 and having the same targetspecificity.

The term “repeat sequence motif” refers to an amino acid sequence, whichis deduced from one or more repeat units or repeat modules. Preferably,said repeat units or repeat modules are from repeat domains havingbinding specificity for the same target. Such repeat sequence motifscomprise framework residue positions and target interaction residuepositions. Said framework residue positions correspond to the positionsof framework residues of the repeat units (or modules). Likewise, saidtarget interaction residue positions correspond to the positions oftarget interaction residues of the repeat units (or modules). Repeatsequence motifs comprise fixed positions and randomized positions. Theterm “fixed position” refers to an amino acid position in a repeatsequence motif, wherein said position is set to a particular amino acid.Most often, such fixed positions correspond to the positions offramework residues and/or the positions of target interaction residuesthat are specific for a certain target. The term “randomized position”refers to an amino acid position in a repeat sequence motif, wherein twoor more amino acids are allowed at said amino acid position, forexample, wherein any of the usual twenty naturally occurring amino acidsare allowed, or wherein most of the twenty naturally occurring aminoacids are allowed, such as amino acids other than cysteine, or aminoacids other than glycine, cysteine and proline. Most often, suchrandomized positions correspond to the positions of target interactionresidues. However, some positions of framework residues may also berandomized.

The term “folding topology” refers to the tertiary structure of saidrepeat units or repeat modules. The folding topology will be determinedby stretches of amino acids forming at least parts of α-helices orδ-sheets, or amino acid stretches forming linear polypeptides or loops,or any combination of α-helices, δ-sheets and/or linearpolypeptides/loops.

The term “consecutive” refers to an arrangement, wherein the repeatunits or repeat modules are arranged in tandem. In designed repeatproteins, there are at least 2, usually about 2 to 6, in particular atleast about 6, frequently 20 or more repeat units (or modules). In mostcases, repeat units (or modules) of a repeat domain will exhibit a highdegree of sequence identity (same amino acid residues at correspondingpositions) or sequence similarity (amino acid residues being different,but having similar physicochemical properties), and some of the aminoacid residues might be key residues being strongly conserved. However, ahigh degree of sequence variability by amino acid insertions and/ordeletions, and/or substitutions between the different repeat units (ormodules) of a repeat domain may be possible as long as the commonfolding topology of the repeat units (or modules) is maintained.

Methods for directly determining the folding topology of repeat proteinsby physicochemical means such as X-ray crystallography, NMR or CDspectroscopy, are well known to the practitioner skilled in the art.Methods for identifying and determining repeat units or repeat sequencemotifs or for identifying families of related proteins comprising suchrepeat units or motifs, such as homology searches (BLAST etc.), are wellestablished in the field of bioinformatics, and are well known to thepractitioner in the art. The step of refining an initial repeat sequencemotif may comprise an iterative process.

The term “repeat modules” refers to the repeated amino acid sequences ofthe designed repeat domains, which are originally derived from therepeat units of naturally occurring repeat proteins. Each repeat modulecomprised in a repeat domain is derived from one or more repeat units ofthe family or subfamily of naturally occurring repeat proteins, e.g. thefamily of armadillo repeat proteins or ankyrin repeat proteins.

“Repeat modules” may comprise positions with amino acid residues presentin all copies of corresponding repeat modules (“fixed positions”) andpositions with differing or “randomized” amino acid residues(“randomized positions”).

A binding protein according to the invention comprises at least oneankyrin repeat domain, wherein said repeat domain has bindingspecificity for mammalian serum albumin (xSA).

The term “has binding specificity for a target”, “specifically bindingto a target” or “target specificity” and the like means that a bindingprotein or binding domain binds in PBS to a target with a lowerdissociation constant than to an unrelated protein such as the E. colimaltose binding protein (MBP). Preferably, the dissociation constant inPBS for the target is at least 10, more preferably 10², even morepreferably 10³, or most preferably 10⁴ times lower than thecorresponding dissociation constant for MBP.

The binding protein of the invention is not an antibody or a fragmentthereof, such as Fab or scFv fragments. Antibodies and fragments thereofare well known to the person skilled in the art.

Also, the binding domain of the invention does not comprise animmunoglobulin fold as present in antibodies and/or the fibronectin typeIII domain. An immunoglobulin fold is a common all-β protein fold thatconsists of a two-layer sandwich of about 7 anti-parallel β-strandsarranged in two β-sheets. Immunoglobulin folds are well known to theperson skilled in the art. For example, such binding domains comprisingan immunoglobulin fold are described in WO 2007/080392 or WO2008/097497.

In particular the invention relates to a binding protein comprising atleast one ankyrin repeat domain, wherein said ankyrin repeat domain hasbinding specificity for a mammalian serum albumin and wherein saidankyrin repeat domain comprises an ankyrin repeat module having an aminoacid sequence selected from the group consisting of SEQ ID NO:49, 50, 51and 52 and sequences, wherein up to 9 amino acids in SEQ ID NO:49, 50,51 and 52 are exchanged by any amino acid.

Preferably, up to 8 amino acids in SEQ ID NO:49, 50, 51 and 52 areexchanged by other amino acid, more preferably up to 7 amino acids, morepreferably up to 6 amino acids, more preferably up to 5 amino acids,even more preferably up to 4 amino acids, more preferably up to 3 aminoacids, more preferably up to 2 amino acids, more preferably up to 1amino acid, and most preferably no amino acid in SEQ ID NO:49, 50, 51and 52 is exchanged.

Preferably, when amino acids are exchanged in SEQ ID NO:49, 50, 51 and52, these amino acids are selected from the group consisting of A, D, E,F, H, I, K, L, M, N, Q, R, S, T, V, W and Y; more preferably from thegroup consisting of A, D, E, H, I, K, L, Q, R, S, T, V, and Y. Alsopreferably, the replacement of amino acids is by a homologous aminoacid; i.e. an amino acid is exchanged by an amino acid having a sidechain with similar biophysical properties. For example, the negativecharged amino acid D may be replaced by the negative charged amino acidE, or a hydrophobic amino acid such as L may be replaced by A, I or V.The replacement of an amino acid by a homologous amino acid is wellknown to the person skilled in the art.

A preferred binding protein comprises at least one ankyrin repeatdomain, wherein said repeat domain binds xSA with a dissociationconstant (Kd) in PBS below 10⁻⁴M. Preferably, said repeat domain bindsxSA with a Kd in PBS below 10⁻⁴M, more preferably below 10⁻⁵M, 10⁻⁶M,10⁻⁷M, or most preferably 10⁻⁸M.

Methods to determine dissociation constants of protein-proteininteractions, such as surface plasmon resonance (SPR) based technologies(e.g. SPR equilibrium analysis) or isothermal titration calorimetry(ITC), are well known to the person skilled in the art. The measured Kdvalues of a particular protein-protein interaction can vary if measuredunder different conditions (e.g., salt concentration, pH). Thus,measurements of Kd values are preferably made with standardizedsolutions of protein and a standardized buffer, such as PBS.

Binding proteins comprising an ankyrin repeat domain binding xSA with aKd in PBS below 10⁻⁴M are shown in the Examples.

An ankyrin repeat domain of a binding protein of the invention bindsxSA. Preferred is a binding protein comprising an ankyrin repeat domainthat binds human serum albumin (HSA).

Further preferred is a binding domain comprising between 70 and 300amino acids, in particular between 100 and 200 amino acids.

Further preferred is a binding protein or binding domain devoid of afree Cys residue. A “free Cys residue” is not involved in the formationof a disulfide bond. Even more preferred is a binding protein or bindingdomain free of any Cys residue.

A binding domain of the invention is an ankyrin repeat domain or adesigned ankyrin repeat domain (Binz et al., 2004, loc. cit.),preferably as described in WO 2002/020565. Examples of designed ankyrinrepeat domains are shown in the Examples.

In a further embodiment, the invention relates to a binding proteincomprising at least one ankyrin repeat domain, wherein said repeatdomain has binding specificity for a mammalian serum albumin and whereinsaid ankyrin repeat domain comprises an amino acid sequence that has atleast 70% amino acid sequence identity with one ankyrin repeat domainselected from the group consisting of SEQ ID NOs: 17 to 31 and 43 to 48,wherein G at position 1 and/or S at position 2 of said ankyrin repeatdomain are optionally missing.

Preferably, such an ankyrin repeat domain in a binding protein of theinvention comprises an amino acid sequence that has at least 70% aminoacid sequence identity with one ankyrin repeat domain selected from thegroup consisting of SEQ ID NO: 21, 27 and 46; preferably 27 and 46. Asdefined above, said ankyrin repeat domain binds xSA with a dissociationconstant (Kd) in PBS below 10⁻⁴M. Preferably, said repeat domain bindsxSA with a Kd in PBS below 10⁻⁴M, more preferably below 10⁻⁵M, 10⁻⁶M,10⁻⁷M, or most preferably 10⁻⁸M.

Preferably, such an ankyrin repeat domain in a binding protein of theinvention comprises an amino acid sequence with at least 70% amino acidsequence identity with “randomized repeat units” or “randomizedpositions” in an ankyrin repeat domain selected from the groupconsisting of SEQ ID NOs: 17 to 31 and 43 to 48.

Preferably, instead of 70% amino acid sequence identity, such an ankyrinrepeat domain in a binding protein of the invention comprises an aminoacid sequence with at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, or most preferredat least 95% amino acid sequence identity.

In a particular embodiment, the binding protein with binding specificityfor mammalian serum albumin defined by replacement of up to 9 aminoacids in ankyrin repeat modules of SEQ ID NO:49, 50, 51 and 52, ordefined by at least 70% amino acid sequence identity with one ankyrinrepeat domain selected from the group consisting of SEQ ID NOs: 17 to 31and 43 to 48, has at least a 5-fold higher terminal plasma half-life ina mammal compared to a corresponding binding protein not binding tomammalian serum albumin, for example the ankyrin repeat domain of SEQ IDNO:32. In such a preferred binding protein the minimum terminal plasmahalf-life in human is at least 1 day, more preferably at least 3 days,even more preferably at least 5 days.

In a further embodiment, the invention relates to a binding protein,wherein said ankyrin repeat domain comprises a repeat module with theankyrin repeat sequence motif

(SEQ ID NO: 53) X₁DX₂X₃X₄X₅TPLHLAAX₆X₇GHLX₈IVEVLLKX₉GADVNA 

wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈ and X₉, represent, independentlyof each other, an amino acid residue selected from the group consistingof A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W and Y;

preferably wherein

X₁ represents an amino acid residue selected from the group consistingof A, D, M, F, S, I,

T, N, Y and K; more preferably of K and A;

X₂ represents an amino acid residue selected from the group consistingof E, K, D, F, M, N, I and Y; more preferably of I, E and Y;

X₃ represents an amino acid residue selected from the group consistingof W, R, N, T, H, K, A and F; more preferably of W, R and F;

X₄ represents an amino acid residue selected from the group consistingof G and S;

X₅ represents an amino acid residue selected from the group consistingof N, T and H;

X₆ represents an amino acid residue selected from the group consistingof N, V and R;

X₇ represents an amino acid residue selected from the group consistingof E, Y, N, D, H, S, A, Q, T and G; more preferably of E, Y and N;

X₈ represents an amino acid residue selected from the group consistingof E and K;

X₉ represents an amino acid residue selected from the group consistingof S, A, Y, H and N; more preferably of Y and H; and

wherein optionally up to 5 amino acids in other than in positionsdenoted with X in SEQ ID NO:53 are exchanged by any amino acid.

In particular, the invention relates to a binding protein, wherein theankyrin repeat domain comprises a repeat module with the ankyrin repeatsequence motif

(SEQ ID NO: 10) X₁DX₂X₃GX₄TPLHLAAX₅X₆GHLEIVEVLLKX₇GADVNA 

wherein

X₁ represents an amino acid residue selected from the group consistingof A, D, M, F, S, I, T, N, Y, and K; preferably of K and A;

X₂ represents an amino acid residue selected from the group consistingof E, K, D, F, M, N, I and Y; preferably of I, E and Y;

X₃ represents an amino acid residue selected from the group consistingof W, R, N, T, H, K, A and F; preferably of W, R and F;

X₄ represents an amino acid residue selected from the group consistingof N, T and H;

X₅ represents an amino acid residue selected from the group consistingof N, V and R;

X₆ represents an amino acid residue selected from the group consistingof E, Y, N, D, H, S, A, Q, T and G; preferably of E, Y and N;

X₇ represent an amino acid residue selected from the group consisting ofS, A, Y, H and N; preferably of Y and H; and

wherein optionally up to 5 amino acids in other than in positionsdenoted with X in SEQ ID NO:10 are exchanged by any amino acid.

In a further embodiment, the invention relates to a binding protein,wherein the ankyrin repeat domain comprises a repeat module with theankyrin repeat sequence motif

(SEQ ID NO: 11) X₁DYFX₂HTPLHLAARX₃X₄HLX₅IVEVLLKX₆GADVNA 

wherein

X₁ represents an amino acid residue selected from the group consistingof D, K and A; preferably K and A;

X₂ represents an amino acid residue selected from the group consistingof D, G and S; preferably G and S;

X₃ represents an amino acid residue selected from the group consistingof E, N, D, H, S, A, Q, T and G; preferably Q, D and N; more preferablyof Q and N;

X₄ represents an amino acid residue selected from the group consistingof G and D;

X₅ represents an amino acid residue selected from the group consistingof E, K and G; preferably E and K;

X₆ represents an amino acid residue selected from the group consistingof H, Y, A and N; preferably H, A and Y; more preferably of A and Y; and

wherein optionally up to 5 amino acids in other than in positionsdenoted with X in SEQ ID NO:11 are exchanged by any amino acid.

In yet another embodiment, the invention relates to a binding protein,wherein the ankyrin repeat domain comprises a repeat module with theankyrin repeat sequence motif

(SEQ ID NO: 54) X₁DFX₂GX₃TPLHLAAX₄X₅GHLEIVEVLLKX₆GADVNA 

wherein

X₁ represents an amino acid residue selected from the group consistingof F, S, L and K; preferably of S and K;

X₂ represents an amino acid residue selected from the group consistingof V and A;

X₃ represents an amino acid residue selected from the group consistingof R and K;

X₄ represents an amino acid residue selected from the group consisting Sand N;

X₅ represents an amino acid residue selected from the group consistingof N, D, Q, S, A, T and E; preferably D and Q;

X₆ represents an amino acid residue selected from the group consistingof A, H, Y, S and N; preferably of A and H; and

wherein optionally up to 5 amino acids in other than in positionsdenoted with X in SEQ ID NO:54 are exchanged by any amino acid.

In particular, the invention relates to a binding protein, wherein theankyrin repeat domain comprises a repeat module with the ankyrin repeatsequence motif

(SEQ ID NO: 12) X₁DFX₂GX₃TPLHLAAX₄DGHLEIVEVLLKX₅GADVNA 

wherein

X₁ represents an amino acid residue selected from the group consistingof F, S, L and K; preferably S and K;

X₂ represents an amino acid residue selected from the group consistingof V and A;

X₃ represents an amino acid residue selected from the group consistingof R and K;

X₄ represents an amino acid residue selected from the group consistingof S and N;

X₅ represents an amino acid residue selected from the group consistingof A, H, Y, S and N; preferably A and H; and

wherein optionally up to 5 amino acids in other than in positionsdenoted with X in SEQ ID NO:12 are exchanged by any amino acid.

Preferred is a binding protein, wherein said ankyrin repeat domaincomprises a repeat module with the ankyrin repeat sequence motif of SEQID NO:12, preceded by a repeat module with the ankyrin repeat sequencemotif of SEQ ID NO:11.

In yet another embodiment, the invention relates to a binding protein,wherein the ankyrin repeat domain comprises a repeat module with theankyrin repeat sequence motif

(SEQ ID NO: 13) X₁DX₂X₃GTTPLHLAAVYGHLEX₄VEVLLKX₅GADVNA 

wherein

X₁ represents an amino acid residue selected from the group consistingof K, A, D, M, F, S, I, T, N, and Y; preferably K and A;

X₂ represents an amino acid residue selected from the group consistingof E, K, D, F, M, N and Y; preferably E, D and Y;

X₃ represents an amino acid residue selected from the group consistingof R, N, T, H, K, A and F; preferably R and F;

X₄ represents an amino acid residue selected from the group consistingof I and M;

X₅ represents an amino acid residue selected from the group consistingof H, Y, K, A and N; preferably K and A; and

wherein optionally up to 5 amino acids in other than in positionsdenoted with X in SEQ ID NO:13 are exchanged by any amino acid.

In yet another embodiment, the invention relates to a binding protein,wherein the ankyrin repeat domain comprises a repeat module with theankyrin repeat sequence motif

(SEQ ID NO: 14) X₁NETGYTPLHLADSSGHX₂EIVEVLLKX₃X₄X₅DX₆NA 

wherein

X₁ represents an amino acid residue selected from the group consistingof Q and K;

X₂ represents an amino acid residue selected from the group consistingof L and P;

X₃ represents an amino acid residue selected from the group consistingof H, N, Y and A; preferably H and A;

X₄ represents an amino acid residue selected from the group consistingof G and S;

X₅ represents an amino acid residue selected from the group consistingof A, V, T and S; preferably S and A;

X₆ represents an amino acid residue selected from the group consistingof V and F; and

wherein optionally up to 5 amino acids in other than in positionsdenoted with X in SEQ ID NO:14 are exchanged by any amino acid.

Preferred is a binding protein, wherein said ankyrin repeat domaincomprises a repeat module with the ankyrin repeat sequence motif of SEQID NO:14, preceded by a repeat module with the ankyrin repeat sequencemotif of SEQ ID NO:13.

The term “capping module” refers to a polypeptide fused to the N- orC-terminal repeat module of a repeat domain, wherein said capping moduleforms tight tertiary interactions (i.e. tertiary structure interactions)with said repeat module thereby providing a cap that shields thehydrophobic core of said repeat module at the side not in contact withthe consecutive repeat module from the solvent. Said N- and/orC-terminal capping module may be, or may be derived from, a capping unitor other structural unit found in a naturally occurring repeat proteinadjacent to a repeat unit. The term “capping unit” refers to a naturallyoccurring folded polypeptide, wherein said polypeptide defines aparticular structural unit which is N- or C-terminally fused to a repeatunit, wherein said polypeptide forms tight tertiary structureinteractions with said repeat unit thereby providing a cap that shieldsthe hydrophobic core of said repeat unit at one side from the solvent.Preferably, capping modules or capping units are capping repeats. Theterm “capping repeat” refers to capping module or capping unit having asimilar or the same fold as said adjacent repeat unit (or module) and/orsequence similarities to said adjacent repeat unit (or module). Cappingmodules and capping repeats are described in WO 2002/020565 and byInterlandi et al., 2008 (loc. cit.). For example, WO 2002/020565describes the N-terminal capping module (i.e. a capping repeat) havingthe amino acid sequence GSDLGKKLLEAARAGQDDEVRILMANGADVNA (SEQ ID NO:1)and

the C-terminal capping module (i.e. a capping repeat) having the aminoacid sequence QDKFGKTAFDISIDNGNEDLAEILQKLN (SEQ ID NO:2).

Interlandi et al., 2008 (loc. cit.) describe the C-terminal cappingmodules having the amino acid sequences QDKFGKTPFDLAIREGHEDIAEVLQKAA(SEQ ID NO:3) and QDKFGKTPFDLAIDNGNEDIAEVLQKAA (SEQ ID NO:4).

For example, the N-terminal capping module of SEQ ID NO:17 is encoded bythe amino acids from position 1 to 32 and the C-terminal capping moduleof SEQ ID NO:17 is encoded by the amino acids form position 99 to 126.

A preferred N-terminal capping module comprises the sequence motif

(SEQ ID NO: 5) X₁LX₂KKLLEAARAGQDDEVRILX₃AX₄GADVNA 

wherein X₁ represents an amino acid residue G, A or D;

wherein X₂ represents an amino acid residue G or D;

wherein X₃ represents an amino acid residue L, V, I, A or M; preferably,L or M; and

wherein X₄ represents an amino acid residue A, H, Y, K, R or N;preferably, A or N.

Further preferred is any such N-terminal capping module comprising anN-terminal capping repeat, wherein one or more of the amino acidsresidues in said capping repeat are replaced by an amino acid residuefound at the corresponding position on alignment of a correspondingcapping unit or repeat unit.

A preferred C-terminal capping module comprises the sequence motif

(SEQ ID NO: 6) X₁DKX₂GKTX₃X₄DX₅X₆X₇DX₈GX₉EDX₁₀AEX₁₁LQKAA 

wherein X₁ represents an amino acid residue Q or K;

wherein X₂ represents an amino acid residue A, S or F; preferably, S orF;

wherein X₃ represents an amino acid residue A or P;

wherein X₄ represents an amino acid residue A or F;

wherein X₅ represents an amino acid residue I or L;

wherein X₆ represents an amino acid residue S or A;

wherein X₇ represents an amino acid residue I or A;

wherein X₈ represents an amino acid residue A, E or N; preferably, A orN;

wherein X₉ represents an amino acid residue N or H;

wherein X₁₀ represents an amino acid residue L or I;

wherein X₁₁ represents an amino acid residue I or V; and

wherein X₂ does not represent F if X₄ represents F and X₇ represents Iand X₈ represents N or E.

A further preferred C-terminal capping module comprises the sequencemotif

(SEQ ID NO: 7) X₁DKX₂GKTX₃ADX₄X₅X₆DX₇GX₈EDX₉AEX₁₀LQKAA 

wherein X₁ represents an amino acid residue Q or K;

wherein X₂ represents an amino acid residue A, S or F; preferably, S orF;

wherein X₃ represents an amino acid residue A or P;

wherein X₄ represents an amino acid residue I or L;

wherein X₅ represents an amino acid residue S or A;

wherein X₆ represents an amino acid residue I or A;

wherein X₇ represents an amino acid residue A, E or N; preferably, A orN;

wherein X₈ represents an amino acid residue N or H;

wherein X₉ represents an amino acid residue L or I; and

wherein X₁₀ represents an amino acid residue I or V.

A further preferred C-terminal capping module comprises the sequencemotif

(SEQ ID NO: 8) X₁DKX₂GKTX₃ADX₄X₅ADX₆GX₇EDX₈AEX₉LQKAA 

wherein X₁ represents an amino acid residue Q or K;

wherein X₂ represents an amino acid residue A, S or F; preferably, S orF;

wherein X₃ represents an amino acid residue A or P;

wherein X₄ represents an amino acid residue I or L;

wherein X₅ represents an amino acid residue S or A;

wherein X₆ represents an amino acid residue A, E or N; preferably, A orN;

wherein X₇ represents an amino acid residue N or H;

wherein X₈ represents an amino acid residue L or I; and

wherein X₉ represents an amino acid residue I or V.

Preferably, such a C-terminal capping module comprising the sequencemotif of SEQ ID NO:6, 7 or 8 has an amino acid residue A, I or K;preferably, I or K; at the position corresponding to position 3 of saidsequence motif.

Also preferably, such a C-terminal capping module comprising thesequence motif of SEQ ID NO:6, 7 or 8 has an amino acid residue R or Dat the position corresponding to position 14 of said sequence motif.

A preferred C-terminal capping module is a C-terminal capping modulehaving the amino acid sequence QDKSGKTPADLAADAGHEDIAEVLQKAA (SEQ IDNO:9).

Further preferred is a C-terminal capping module having the amino acidsequence of SEQ ID NO:9, wherein

the amino acid residue at position 1 is Q or K;

the amino acid residue at position 4 is S or F;

the amino acid residue at position 9 is A or F;

the amino acid residue at position 13 is A or I;

the amino acid residue at position 15 is A, E or N; and

wherein said C-terminal capping module has not the amino acid sequenceof SEQ ID NO:2, 3 or 4.

Further preferred is a C-terminal capping module having an amino acidsequence comprising at least 70%, preferably at least 75%, 80%, 85%,90%, or most preferred at least 95% amino acid sequence identity onalignment with SEQ ID NO:9 or 2. Preferably, the amino acid residue ofsaid C-terminal capping module at the position corresponding to position4 of SEQ ID:9 on alignment is S, the amino acid residue of saidC-terminal capping module at the position corresponding to position 9 ofSEQ ID:9 on alignment is A, the amino acid residue of said C-terminalcapping module at the position corresponding to position 13 of SEQ ID:9on alignment is A, and/or the amino acid residue of said C-terminalcapping module at the position corresponding to position 15 of SEQ ID:9on alignment is A. Further preferably, the amino acid residue of saidC-terminal capping module at the position corresponding to position 9 ofSEQ ID:9 on alignment is A and/or the amino acid residue of saidC-terminal capping module at the position corresponding to position 13of SEQ ID:9 on alignment is A. Also preferably, said C-terminal cappingmodule comprises 28 amino acids.

Further preferred is a C-terminal capping module having the amino acidsequence of SEQ ID NO:2 or 9, wherein

one or more of the amino acid residues of said C-terminal capping moduleare exchanged by an amino acid found at the corresponding position onalignment of a corresponding C-terminal capping repeat or capping unitand wherein the amino acid residue at position 4 is S;

the amino acid residue at position 9 is A;

the amino acid residue at position 13 is A; and/or

the amino acid residue at position 15 is A.

Preferably, up to 30% of the amino acid residues of said C-terminalcapping module are exchanged, more preferably, up to 20% and even morepreferably, up to 10% of the amino acid residues are exchanged. Alsopreferably, such a C-terminal capping module is a naturally occurringC-terminal capping repeat.

Also preferred is a C-terminal capping module comprising the amino acidsfrom position 1 to 25 or from position 1 to 26 of any of the aboveC-terminal capping modules based on SEQ ID NO:9.

Further preferred is such a C-terminal capping module having an aminoacid sequence not comprising the amino acid N followed by G.

Also preferred is a C-terminal capping module having an at least 70%,preferably at least 75%, 80%, 85%, 90%, or most preferred at least 95%amino acid sequence identity with any of the above C-terminal cappingmodules based on SEQ ID NO:9 or with SEQ ID NO:9 itself.

Further preferred is a C-terminal capping module having an at least 70%,preferably at least 75%, 80%, 85%, 90%, or most preferred at least 95%amino acid sequence identity with SEQ ID NO:2 or 9 and wherein saidC-terminal capping module has amino acid A at position 9; preferably,said C-terminal capping module has amino acid A at positions 9 and 13;more preferably, said C-terminal capping module has amino acid A atpositions 9, 13 and 15; and most preferably, said C-terminal cappingmodule has amino acid A at positions 9, 13 and 15 and S at position 4.

Further preferred is such a C-terminal capping module not having theamino acid R at position 14 and/or not having the amino acid E atposition 15.

Also preferred is such an C-terminal capping module not having an aminoacid sequence identical to SEQ ID NO:2, 3 or 4.

Further preferred is such a C-terminal capping module having an aminoacid sequence based on SEQ ID NO:9, wherein said C-terminal cappingmodule has amino acids at positions 26, 27 and 28 selected from thegroup consisting of A, L, R, M, K and N; more preferably, A, L, R and K;and most preferably, K, A and L.

A capping module of a repeat domain can be exchanged by a capping moduleof the invention by combining techniques, such as alignment of aminoacid sequences, mutagenesis and gene synthesis, known to the personskilled in the art. For example, the C-terminal capping repeat of SEQ IDNO:17 can be replaced by C-terminal capping repeat of SEQ ID NO:9 by (i)determination of the C-terminal capping repeat of SEQ ID NO:17 (i.e.sequence position 99 to 126) by sequence alignment with SEQ ID NO:9,(ii) replacing the sequence of the determined C-terminal capping repeatof SEQ ID NO:17 with the sequence of SEQ ID NO:9, (iii) generation of agene encoding the repeat domain encoding the exchanged C-terminalcapping module, (iv) expressing of the modified repeat domain in thecytoplasm of E. coli and (v) purification of the modified repeat domainby standard means.

Furthermore, a repeat domain of the invention can be constructedgenetically by assembling a N-terminal capping module (i.e. theN-terminal capping repeat of SEQ ID NO:1) followed by one or more repeatmodules (i.e. the repeat modules comprising the amino acid residues fromposition 33 to 98 of SEQ ID NO:17) and a C-terminal capping module (i.e.the C-terminal capping repeat of SEQ ID NO:9) by means of genesynthesis. The genetically assembled repeat domain gene can then beexpressed in E. coli as described above.

Also preferred is a binding protein, wherein the ankyrin repeat domainor designed ankyrin repeat domain comprises a C-terminal capping modulewith the sequence motif of SEQ ID NO:6, 7 or 8, wherein said cappingmodule has the amino acid I at position 3 and wherein said repeat moduleis preceded by a repeat module with the ankyrin repeat sequence motif ofSEQ ID NO:12.

Further preferred is a binding protein, a repeat domain, an N-terminalcapping module or a C-terminal capping module having an amino acidsequence devoid of amino acids C, M or N.

Further preferred is a binding protein, a repeat domain, an N-terminalcapping module or a C-terminal capping module having an amino acidsequence devoid of amino acid N followed by G.

Further preferred is any such C-terminal capping module comprising aC-terminal capping repeat, wherein one or more of the amino acidsresidues in said capping repeat are replaced by an amino acid residuefound at the corresponding position on alignment of a correspondingcapping unit or repeat unit.

Further preferred is a binding protein comprising any such N-terminal orC-terminal capping module.

Examples of amino acid sequences of such C-terminal capping modules arethe amino acid sequences from position 99 to 126 in SEQ ID NOs:19, 21,27, 28, 38, 40 and 42. Example 6 demonstrates that the thermal stabilityof a repeat domain can be increased by replacing their C-terminalcapping modules by a capping module of the invention.

The term “target” refers to an individual molecule such as a nucleicacid molecule, a polypeptide or protein, a carbohydrate, or any othernaturally occurring molecule, including any part of such individualmolecule, or complexes of two or more of such molecules. The target maybe a whole cell or a tissue sample, or it may be any non-naturalmolecule or moiety. Preferably, the target is a naturally occurring ornon-natural polypeptide or a polypeptide containing chemicalmodifications, for example modified by natural or non-naturalphosphorylation, acetylation, or methylation. In the particularapplication of the present invention, the target is xSA.

The term “xSA” refers to a mammalian serum albumin, such as a serumalbumin from mouse, rat, rabbit, dog, pig, monkey or human. The term“MSA” refers to a mouse serum albumin (UniProtKB/Swiss-Prot primaryaccession number P07724), the term “CSA” refers to a cynomolgus monkey(i.e. macaca fascicularis) serum albumin (UniProtKB/Swiss-Prot primaryaccession number A2V9Z4) and the term “HSA” refers to a human serumalbumin (UniProtKB/Swiss-Prot primary accession number P02768).

The term “consensus sequence” refers to an amino acid sequence, whereinsaid consensus sequence is obtained by structural and/or sequencealigning of multiple repeat units. Using two or more structural and/orsequence aligned repeat units, and allowing for gaps in the alignment,it is possible to determine the most frequent amino acid residue at eachposition. The consensus sequence is that sequence which comprises theamino acids which are most frequently represented at each position. Inthe event that two or more amino acids are represented above-average ata single position, the consensus sequence may include a subset of thoseamino acids. Said two or more repeat units may be taken from the repeatunits comprised in a single repeat protein, or from two or moredifferent repeat proteins.

Consensus sequences and methods to determine them are well known to theperson skilled in the art.

A “consensus amino acid residue” is the amino acid found at a certainposition in a consensus sequence. If two or more, e.g. three, four orfive, amino acid residues are found with a similar probability in saidtwo or more repeat units, the consensus amino acid may be one of themost frequently found amino acids or a combination of said two or moreamino acid residues.

Further preferred are non-naturally occurring capping modules, repeatmodules, binding proteins or binding domains.

The term “non-naturally occurring” means synthetic or not from nature,more specifically, the term means made from the hand of man. The term“non-naturally occurring binding protein” or “non-naturally occurringbinding domain” means that said binding protein or said binding domainis synthetic (i.e. produced by chemical synthesis from amino acids) orrecombinant and not from nature. “Non-naturally occurring bindingprotein” or “non-naturally occurring binding domain” is a man-madeprotein or domain, respectively, obtained by expression ofcorrespondingly designed nucleic acids. Preferably, the expression isdone in eukaryotic or bacterial cells, or by using a cell-free in vitroexpression system. Further, the term means that the sequence of saidbinding protein or said binding domain is not present as anon-artificial sequence entry in a sequence database, for example inGenBank, EMBL-Bank or Swiss-Prot. These databases and other similarsequence databases are well known to the person skilled in the art.

The invention relates to a binding protein comprising a binding domain,wherein said binding domain is an ankyrin repeat domain and specificallybinds to xSA and wherein said binding protein and/or binding domain hasa midpoint denaturation temperature (Tm) above 40° C. upon thermalunfolding in PBS and forms less than 5% (w/w) insoluble aggregates atconcentrations up to 10 g/L when incubated at 37° C. for 1 day in PBS.

The term “PBS” means a phosphate buffered water solution containing 137mM NaCl, 10 mM phosphate and 2.7 mM KCl and having a pH of 7.4.

Preferably, the binding protein and/or binding domain has a midpointdenaturation temperature (Tm) above 45° C., more preferably above 50°C., more preferably above 55° C., and most preferably above 60° C. uponthermal unfolding in PBS at pH 7.4 or in MES buffer at pH 5.8. A bindingprotein or a binding domain of the invention possesses a definedsecondary and tertiary structure under physiological conditions. Thermalunfolding of such a polypeptide results in a loss of its tertiary andsecondary structure, which can be followed, for example, by circulardichroism (CD) measurements. The midpoint denaturation temperature of abinding protein or binding domain upon thermal unfolding corresponds tothe temperature at the midpoint of the cooperative transition inphysiological buffer upon heat denaturation of said protein or domain byslowly increasing the temperature from 10° C. to about 100° C. Thedetermination of a midpoint denaturation temperature upon thermalunfolding is well known to the person skilled in the art. This midpointdenaturation temperature of a binding protein or binding domain uponthermal unfolding is indicative of the thermal stability of saidpolypeptide.

Also preferred is a binding protein and/or binding domain forming lessthan 5% (w/w) insoluble aggregates at concentrations up to 20 g/L,preferably up 40 g/L, more preferably up to 60 g/L, even more preferablyup to 80 g/L, and most preferably up to 100 g/L when incubated for over5 days, preferably over 10 days, more preferably over 20 days, morepreferably over 40 days, and most preferably over 100 days at 37° C. inPBS. The formation of insoluble aggregates can be detected by theappearance of visual precipitations, gel filtration or dynamic lightscattering, which strongly increases upon formation of insolubleaggregates. Insoluble aggregates can be removed from a protein sample bycentrifugation at 10'000×g for 10 minutes. Preferably, a binding proteinand/or binding domain forms less than 2%, more preferably less than 1%,0.5%, 0.2%, 0.1%, or most preferably less than 0.05% (w/w) insolubleaggregates under the mentioned incubation conditions at 37° C. in PBS.Percentages of insoluble aggregates can be determined by separation ofthe insoluble aggregates from soluble protein, followed by determinationof the protein amounts in the soluble and insoluble fraction by standardquantification methods.

Also preferred is a binding protein and/or binding domain that does notlose its native three-dimensional structure upon incubation in PBScontaining 100 mM dithiothreitol (DTT) for 1 or 10 hours at 37° C.

In one particular embodiment the invention relates to a binding proteincomprising a binding domain being an ankyrin repeat domain, specificallybinding to xSA and having the indicated or preferred midpointdenaturation temperature and non-aggregating properties as definedabove, wherein said binding protein has an at least 5-fold higherterminal plasma half-life in a mammal compared to a binding domain notbinding to a serum protein such as xSA.

Preferably, said binding domain has an at least 10-fold, more preferablyat least 20-fold, 40-fold, 100-fold, 300-fold, or most preferably atleast 10³-fold higher terminal plasma half-life in a mammal compared toa binding domain not binding to a serum protein such as xSA.

Also preferably, said binding domain does not bind xSA indicated by a Kdabove 10⁻⁴M, more preferably above 10⁻³M or most preferably above 10⁻²Mfor binding of xSA. An example of a binding domain that does not bindxSA is the repeat domain of SEQ ID NO:32.

Further preferably, said binding domain is a repeat domain and has an atleast 5-fold, more preferably at least 10-fold, 20-fold, 40-fold,100-fold, 300-fold, or most preferably at least 10³-fold higher (i.e.longer) terminal plasma half-life in a mammal compared to the repeatdomain of SEQ ID NO:32 or compared to DARPin #32, DARPin #41 or DARPin#42.

A preferred binding protein comprises a binding domain with bindingspecificity for HSA having a terminal plasma half-life of above 1, morepreferably above 3, 5, 7, 10, 15, or most preferably above 20 days inhumans.

The terminal plasma half-life of a binding domain can be determined byassays well know to the person skilled in the art (Toutain, P. L., andBousquet-Mélou, A., J. Vet. Pharmacol. Ther. 27(5), 427-439, 2004).Examples on the determination of terminal plasma half-life are given inthe Examples.

The term “terminal plasma half-life” of a drug such as a binding proteinor binding domain of the invention refers to the time required to reachhalf the plasma concentration of the drug applied to a mammal afterreaching pseudo-equilibrium. This half-life is not defined as the timerequired to eliminate half the dose of the drug administered to themammal.

In one particular embodiment the invention relates to a binding proteincomprising a binding domain being an ankyrin repeat domain, specificallybinding to xSA and comprising a bioactive compound.

The term “bioactive compound” refers to a compound that is diseasemodifying when applied to a mammal having said disease. A bioactivecompound may have antagonistic or agonistic properties and can be aproteinaceous bioactive compound or a non-proteinaceous bioactivecompound.

Such proteinaceous bioactive compounds can be covalently attached to,for example, a binding domain of the invention by the generation ofgenetic fusion polypeptides using standard DNA cloning technologies,followed by their standard expression and purification. For example,DARPin #36 comprises a repeat domain with binding specificity for ahuman growth factor (i.e. a bioactive compound) followed by a repeatdomain with binding specificity for HSA.

Such non-proteinaceous bioactive compounds can be covalently attachedto, for example, a binding domain of the invention by chemical means,e.g., by coupling to a cysteine thiol via a maleimide linker with acysteine being coupled via a peptide linker to the N- or C-terminus of abinding domain as described herein.

Examples of proteinaceous bioactive compounds are binding domains havinga distinct target specificity (e.g. neutralizing a growth factor bybinding to it), cytokines (e.g. interleukins), growth factors (e.g.human growth hormone), antibodies and fragments thereof, hormones (e.g.GLP-1) and any possible proteinaceous drug.

Examples of non-proteinaceous bioactive compounds are, toxins (e.g. DM1from ImmunoGen), small molecules targeting GPCRs, antibiotics and anypossible non-proteinaceous drug.

In one particular embodiment the invention relates to a binding proteincomprising an ankyrin repeat domain specifically binding to xSA andfurther comprising a bioactive compound, wherein said binding proteinhas an at least 2-fold higher terminal half-life in a mammal compared tothe terminal half-life of said unmodified bioactive compound, whereinsaid higher terminal half-life is conferred to said binding protein bysaid repeat domain.

Preferably, said binding protein has an at least 5-fold, more preferablyat least 10-fold, 20-fold, 40-fold, 100-fold, 300-fold, or mostpreferably at least 10³-fold higher terminal plasma half-life in amammal compared to said unmodified bioactive compound.

Another preferred embodiment is a recombinant binding protein comprisinga binding domain specifically binding to xSA and wherein said bindingdomain is an ankyrin repeat domain or a designed ankyrin repeat domain.Such an ankyrin repeat domain may comprise one, two, three or moreinternal repeat modules that will participate in binding to xSA.Preferably, such an ankyrin repeat domain comprises an N-terminalcapping module, two to four internal repeat modules, and a C-terminalcapping module. Preferably, said binding domain is an ankyrin repeatdomain or designed ankyrin repeat domain. Also preferably, said cappingmodules are capping repeats.

In particular, the invention relates to a binding protein as definedherein above, wherein the ankyrin repeat domain competes for binding toa mammalian serum albumin with an ankyrin repeat domain selected fromthe group consisting of SEQ ID NOs: 17 to 31 and 43 to 48; preferablySEQ ID NOs: 17 to 31; more preferably SEQ ID NO:19, 21, 27 and 28, inparticular SEQ ID NO:19 and 27.

Most preferred is a binding protein, wherein the ankyrin repeat domainis selected from the group consisting of SEQ ID NOs: 17 to 31 and 43 to48, wherein G at position 1 and/or S at position 2 of said ankyrinrepeat domain are optionally missing.

Also preferably said repeat domain competes for binding to xSA with abinding protein selected from the group of DARPins #17 to 31 and 43 to48. Preferably, said repeat domain competes for binding to xSA with abinding protein from the group of DARPins #19, 21, 27, 28, 45, 46, 47and 48. More preferably, said repeat domain competes for binding to xSAwith binding protein DARPin #19, 45, 46, 48 or 27; even more preferably,said repeat domain competes for binding to xSA with the binding proteinDARPin #46 or 27.

The term “compete for binding” means the inability of two differentbinding domains of the invention to bind simultaneously to the sametarget, while both are able to bind the same target individually. Thus,such two binding domains compete for binding to said target. Preferably,said two competing binding domains bind to an overlapping or the samebinding epitope on said target. Methods, such as competitionEnzyme-Linked Immuno Sorbent Assay (ELISA) or competition SPRmeasurements (e.g. by using the Proteon instrument from BioRad), todetermine if two binding domains compete for binding to a target, arewell known to the practitioner in the art.

Another preferred embodiment is a binding protein comprising a repeatdomain with binding specificity for xSA selected from the groupconsisting of the repeat domains of SEQ ID NO:17 to 31. Preferably, saidrepeat domain is the repeat domain of SEQ ID NO:19, 21, 27 or 28. Morepreferably, said repeat domain is the repeat domain of SEQ ID NO:19.Also more preferably, said repeat domain is the repeat domain of SEQ IDNO:21. Also more preferably, said repeat domain is the repeat domain ofSEQ ID NO:27. Also more preferably, said repeat domain is the repeatdomain of SEQ ID NO:28.

Further preferred is a binding protein, wherein said repeat domain withbinding specificity for xSA comprises an amino acid sequence that has atleast 70% amino acid sequence identity with a repeat domain of saidgroup of repeat domains. Preferably, said amino acid sequence identityis at least 75%, more preferably at least 80%, more preferably at least85%, more preferably at least 90%, and most preferably at least 95%.

Further preferred is a binding protein, wherein said repeat domain withbinding specificity for xSA comprises a repeat module that has at least70% amino acid sequence identity with a repeat module of a repeat domainof said group of repeat domains. Preferably, said amino acid sequenceidentity is at least 75%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, and most preferably at least95%.

Further preferred is a binding protein, wherein said binding proteincomprises two or more of said repeat domains with binding specificityfor xSA. Preferably, said binding protein comprises 2 or 3 of saidrepeat domains. Said two or more repeat domains have the same ordifferent amino acid sequence.

In a further preferred embodiment of a binding protein comprising arepeat domain according to the present invention, one or more of theamino acid residues of the repeat modules of said repeat domain areexchanged by an amino acid residue found at the corresponding positionon alignment of a repeat unit. Preferably, up to 30% of the amino acidresidues are exchanged, more preferably, up to 20%, and even morepreferably, up to 10% of the amino acid residues are exchanged. Mostpreferably, such a repeat unit is a naturally occurring repeat unit.

In a further preferred embodiment of a binding protein comprising arepeat domain according to the present invention, one or more of theamino acid residues of the N-terminal capping module of said repeatdomain is exchanged by an amino acid residue found at the correspondingposition on alignment of an N-terminal capping unit. Preferably, up to30% of the amino acid residues are exchanged, more preferably, up to20%, and even more preferably, up to 10% of the amino acid residues areexchanged. Most preferably, such an N-terminal capping unit is anaturally occurring N-terminal capping unit.

In a further preferred embodiment of a binding protein comprising arepeat domain according to the present invention, one or more of theamino acid residues of the C-terminal capping module of said repeatdomain is exchanged by an amino acid residue found at the correspondingposition on alignment of a C-terminal capping unit. Preferably, up to30% of the amino acid residues are exchanged, more preferably, up to20%, and even more preferably, up to 10% of the amino acid residues areexchanged. Most preferably, such a C-terminal capping unit is anaturally occurring C-terminal capping unit.

In still another particular embodiment, up to 30% of the amino acidresidues, more preferably, up to 20%, and even more preferably, up to10% of the amino acid residues are exchanged with amino acids which arenot found in the corresponding positions of repeat units, N-terminalcapping units or C-terminal capping units.

In further embodiments, any of the xSA binding proteins or domainsdescribed herein may be covalently bound to one or more additionalmoieties, including, for example, a moiety that binds to a differenttarget to create a dual-specificity binding agent, a bioactive compound,a labeling moiety (e.g. a fluorescent label such as fluorescein, or aradioactive tracer), a moiety that facilitates protein purification(e.g. a small peptide tag, such as a His- or strep-tag), a moiety thatprovides effector functions for improved therapeutic efficacy (e.g. theFc part of an antibody to provide antibody-dependent cell-mediatedcytotoxicity, a toxic protein moiety such as Pseudomonas aeruginosaexotoxin A (ETA) or a small molecular toxic agent such as maytansinoidsor DNA alkylating agents) or a moiety that provides improvedpharmacokinetics. Improved pharmacokinetics may be assessed according tothe perceived therapeutic need. Often it is desirable to increasebioavailability and/or increase the time between doses, possibly byincreasing the time that a protein remains available in the serum afterdosing. In some instances, it is desirable to improve the continuity ofthe serum concentration of the protein over time (e.g., decrease thedifference in serum concentration of the protein between theconcentration shortly after administration and the concentration shortlybefore the next administration).

In a further embodiment, the invention relates to nucleic acid moleculesencoding the particular binding proteins, the particular N-terminalcapping modules or the particular C-terminal capping modules. Further, avector comprising said nucleic acid molecule is considered.

Further, a pharmaceutical composition comprising one or more of theabove mentioned binding proteins, in particular binding proteinscomprising repeat domains, or nucleic acid molecules encoding theparticular binding proteins, and optionally a pharmaceutical acceptablecarrier and/or diluent is considered. Pharmaceutical acceptable carriersand/or diluents are known to the person skilled in the art and areexplained in more detail below. Even further, a diagnostic compositioncomprising one or more of the above mentioned binding proteins, inparticular binding proteins comprising repeat domains, is considered.

A pharmaceutical composition comprises binding proteins as describedabove and a pharmaceutically acceptable carrier, excipient orstabilizer, for example as described in Remington's PharmaceuticalSciences 16^(th) edition, Osol, A. Ed. [1980]. Suitable carriers,excipients or stabilizers known to the skilled man are saline, Ringer'ssolution, dextrose solution, Hank's solution, fixed oils, ethyl oleate,5% dextrose in saline, substances that enhance isotonicity and chemicalstability, buffers and preservatives. Other suitable carriers includeany carrier that does not itself induce the production of antibodiesharmful to the individual receiving the composition such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids and amino acid copolymers. A pharmaceutical composition may alsobe a combination formulation, comprising an additional active agent,such as an anti-cancer agent or an anti-angiogenic agent.

The formulations to be used for in vivo administration must be asepticor sterile. This is readily accomplished by filtration through sterilefiltration membranes.

The pharmaceutical composition may be administered by any suitablemethod within the knowledge of the skilled man. The preferred route ofadministration is parenterally. In parenteral administration, themedicament of this invention will be formulated in a unit dosageinjectable form such as a solution, suspension or emulsion, inassociation with the pharmaceutically acceptable excipients as definedabove. The dosage and mode of administration will depend on theindividual to be treated and the particular disease. Generally, thepharmaceutical composition is administered so that the binding proteinof the present invention is given at a dose between 1 μg/kg and 20mg/kg, more preferably between 10 μg/kg and 5 mg/kg, most preferablybetween 0.1 and 2 mg/kg. Preferably, it is given as a bolus dose.Continuous infusion may also be used and includes continuoussubcutaneous delivery via an osmotic minipump. If so, the pharmaceuticalcomposition may be infused at a dose between 5 and 20 μg/kg/minute, morepreferably between 7 and 15 μg/kg/minute.

Further, any of the above mentioned pharmaceutical composition isconsidered for the treatment of a disorder.

The invention further provides methods of treatment. The methodcomprises administering, to a patient in need thereof, a therapeuticallyeffective amount of a binding protein of the invention.

Further, a method of treating a pathological condition in a mammalincluding man, comprising administering to a patient in need thereof aneffective amount of the above mentioned pharmaceutical composition isconsidered.

The binding protein according to the invention may be obtained and/orfurther evolved by several methods such as display on the surface ofbacteriophages (WO 1990/002809, WO 2007/006665) or bacterial cells (WO1993/010214), ribosomal display (WO 1998/048008), display on plasmids(WO 1993/008278) or by using covalent RNA-repeat protein hybridconstructs (WO 2000/032823), or intracellular expression andselection/screening such as by protein complementation assay (WO1998/341120). Such methods are known to the person skilled in the art.

A library of ankyrin repeat proteins used for the selection/screening ofa binding protein according to the invention may be obtained accordingto protocols known to the person skilled in the art (WO 2002/020565,Binz, H. K., et al., J. Mol. Biol., 332, 489-503, 2003, and Binz et al.,2004, loc. cit). The use of such a library for the selection xSAspecific DARPins is given in Example 1. In analogy, the ankyrin repeatsequence motifs as presented above can be used to build libraries ofankyrin repeat proteins that may be used for the selection or screeningof xSA specific DARPins. Furthermore, repeat domains of the presentinvention may be modularly assembled from repeat modules according tothe current invention and appropriate capping modules or capping repeats(Forrer, P., et al., FEBS letters 539, 2-6, 2003) using standardrecombinant DNA technologies (e.g. WO 2002/020565, Binz et al., 2003,loc. cit. and Binz et al., 2004, loc. cit).

The invention is not restricted to the particular embodiments describedin the Examples. Other sources may be used and processed following thegeneral outline described below.

EXAMPLES

All of the starting materials and reagents disclosed below are known tothose skilled in the art, and are available commercially or can beprepared using well-known techniques.

Materials

Chemicals were purchased from Fluka (Switzerland). Oligonucleotides werefrom Microsynth (Switzerland). Unless stated otherwise, DNA polymerases,restriction enzymes and buffers were from New England Biolabs (USA) orFermentas (Lithuania). The cloning and protein production strain was E.coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA). Purified serumalbumin and sera from different species were purchased (e.g. fromSigma-Aldrich, Switzerland or Innovative Research, USA). Biotinylatedserum albumin of different species was obtained chemically via couplingof the biotin moiety to primary amines of the purified serum albuminsusing standard biotinylation reagents and methods (Pierce, USA).

Molecular Biology

Unless stated otherwise, methods are performed according to describedprotocols (Sambrook J., Fritsch E. F. and Maniatis T., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory 1989, NewYork).

Designed Ankyrin Repeat Protein Libraries

The N2C and N3C designed ankyrin repeat protein libraries are described(WO 2002/020565; Binz et al. 2003, loc. cit.; Binz et al. 2004, loc.cit.). The digit in N2C and N3C describes the number of randomizedrepeat modules present between the N-terminal and C-terminal cappingmodules. The nomenclature used to define the positions inside the repeatunits and modules is based on Binz et al. 2004, loc. cit. with themodification that borders of the ankyrin repeat modules and ankyrinrepeat units are shifted by one amino acid position. For example,position 1 of an ankyrin repeat module of Binz et al. 2004 (loc. cit.)corresponds to position 2 of a ankyrin repeat module of the currentdisclosure and consequently position 33 of a ankyrin repeat module ofBinz et al. 2004, loc. cit. corresponds to position 1 of a followingankyrin repeat module of the current disclosure.

All the DNA sequences were confirmed by sequencing, and the calculatedmolecular weight of all described proteins was confirmed by massspectrometry.

Example 1 Selection of Binding Proteins Comprising a Repeat Domain withBinding Specificity for xSA

Using ribosome display (Hanes, J. and Plückthun, A., PNAS 94, 4937-42,1997) many designed ankyrin repeat proteins (DARPins) with bindingspecificity for xSA were selected from the N2C or N3C DARPin librariesdescribed by Binz et al. 2004 (loc. cit.). The binding of the selectedclones toward specific (xSA; i.e. MSA, HSA or CSA) and unspecific (MBP,E. coli maltose binding protein) targets was assessed by crude extractELISA indicating that xSA binding proteins were successfully selected.The repeat domains of SEQ ID NO:17 to 31 constitute amino acid sequencesof selected binding proteins comprising a repeat domain with bindingspecificity for xSA. Sequence analysis of selected binders revealedspecific ankyrin repeat sequence motifs inherent to certain selectedfamilies of binders. Such ankyrin repeat sequence motifs present inrepeat domains with binding specificity for xSA are provided in SEQ IDNO:11 to 14.

Selection of Serum Albumin Specific Ankyrin Repeat Proteins by RibosomeDisplay

The selection of serum albumin specific ankyrin repeat proteins wasperformed by ribosome display (Hanes and Plückthun, loc. cit.) usingHSA, CSA or MSA as target proteins, the library of designed ankyrinrepeat proteins as described (WO 2002/020565, Binz et al., 2003, loc.cit. and Binz et al., 2004, loc. cit) and established protocols (Zahnd,C., Amstutz, P. and Plückthun, A., Nat. Methods 4, 69-79, 2007).Ribosome-display selection rounds were performed on HSA, CSA or MSA(including biotinylated variants of HSA or MSA immobilized overneutravidin or streptavidin) with both the N2C and N3C DARPin librariesusing established protocols (Binz et al. 2004, loc. cit.). The number ofreverse transcription (RT)-PCR cycles after each selection round wasconstantly reduced from 40 to 30, adjusting to the yield due toenrichment of binders. Four selection rounds on HSA, CSA or MSA yieldedpools of micromolar to nanomolar-affinity DARPins, as revealed by ELISAand SPR measurements of single clones. The affinity of certain DARPinswas further improved by using affinity maturation by methods well knownto the person skilled in the art (e.g. by diversifying of DARPin clonesby error prone PCR and selection and screening for improved binders asdescribed above).

Selected Clones Bind Specifically to Serum Albumin as Shown by CrudeExtract ELISA

Individual selected DARPins specifically binding xSA were identified byan enzyme-linked immunosorbent assay (ELISA) using crude Escherichiacoli extracts of DARPin expression cells using standard protocols. Byribosome display selected clones were cloned into the pQE30 (Qiagen)expression vector, transformed into E. coli XL1-Blue (Stratagene) andthen grown overnight at 37° C. in a 96-deep-well plate (each clone in asingle well) containing 1 ml growth medium (2YT containing 1% glucoseand 100 μg/ml ampicillin). 1 ml of fresh 2YT containing 50 μg/mlampicillin was inoculated with 100 μl of the overnight culture in afresh 96-deep-well plate. After incubation for 2 h at 37° C., expressionwas induced with IPTG (1 mM final concentration) and continued for 3 h.Cells were harvested, resuspended in 100 μl B-PERII (Pierce) andincubated for 15 min at room temperature with shaking. Then, 900 μlPBS-TC (PBS supplemented with 0.25% Casein hydrolysate, 0.1% Tween 20®,pH 7.4) were added and cell debris were removed by centrifugation. 100μl of each lysed clone were applied to a well of a NeutrAvidin coatedMaxiSorp plate containing either xSA or the unrelated MBP immobilizedvia their biotin moiety and incubated for 1 h at RT. After extensivewashing with PBS-T (PBS supplemented with 0.1% Tween 20®, pH 7.4) theplate was developed using standard ELISA procedures using the monoclonalanti-RGS(His)₄ antibody (34650, Qiagen) as primary antibody and apolyclonal goat anti-mouse antibody conjugated with alkaline phosphatase(A3562, Sigma) as secondary reagent. Binding was then detected by usingdisodium 4-nitrophenyl phosphate (4NPP, Fluka) as a substrate foralkaline phosphatase. The color development was measured at 405 nm.Screening of several hundred clones by such a crude cell extract ELISArevealed more than hundred different DARPins with specificity for xSA.These binding proteins were chosen for further analysis. Examples ofamino acid sequences of selected repeat domains that specifically bindto xSA are provided in SEQ ID NO:17 to 31, 37 to 40, and 43 to 48.

Deducing Repeat Sequence Motives from Selected Repeat Domains withBinding Specificity for xSA

The amino acid sequences of selected repeat domains with bindingspecificity for xSA were further analyzed by sequence analyzing toolsknown to the practitioner in the art (WO 2002/020565; Forrer et al.,2003, loc. cit.; Forrer, P., Binz, H. K., Stumpp, M. T. and Plückthun,A., ChemBioChem, 5(2), 183-189, 2004). Nevertheless, in contrast to WO2002/020565 where naturally occurring repeat motifs were used to deducerepeat sequence motifs, here the repeat sequence motifs were deducedfrom the repeat units of selected repeat domains with bindingspecificity for xSA. Thereby families of selected repeat domainscomprising a common repeat sequence motif were determined. Such repeatsequence motifs present in repeat domains with binding specificity forxSA are provided in SEQ ID NO:11 to 14.

High Level and Soluble Expression of DARPins

For further analysis, the selected clones showing specific xSA bindingin the crude cell extract ELISA as described above were expressed in E.coli BL21 or XL1-Blue cells and purified using their His-tag usingstandard protocols. 25 ml of stationary overnight cultures (LB, 1%glucose, 100 mg/l of ampicillin; 37° C.) were used to inoculate 1 lcultures (same medium). At an absorbance of 0.7 at 600 nm, the cultureswere induced with 0.5 mM IPTG and incubated at 37° C. for 4 h. Thecultures were centrifuged and the resulting pellets were resuspended in40 ml of TBS500 (50 mM Tris-HCl, 500 mM NaCl, pH 8) and sonicated. Thelysate was recentrifuged, and glycerol (10% (v/v) final concentration)and imidazole (20 mM final concentration) were added to the resultingsupernatant. Proteins were purified over a Ni-nitrilotriacetic acidcolumn (2.5 ml column volume) according to the manufacturer'sinstructions (QIAgen, Germany). Alternatively, DARPins or selectedrepeat domains devoid of a 6×His-tag were purified by anion exchangechromatography followed by size exclusion chromatography according tostandard resins and protocols known to the person skilled in the art. Upto 200 mg of highly soluble DARPins with binding specificity to serumalbumin can be purified from one liter of E. coli culture with apurity >95% as estimated from SDS-15% PAGE. Such purified DARPins areused for further characterizations.

Example 2 Stability Analysis and Size Exclusion Chromatography ofDARPins with Binding Specificity for xSA

DARPins #19 to 22 and DARPins #27 to 30 with binding specificity for xSAwere purified to near homogeneity using their His-tag as described aboveand stored in PBS for 28 days at 30 mg/ml (˜2 mM) at 40° C. (stabilitystudy). At day 0 (FIG. 1A) and day 28 (FIG. 1B) samples were taken,diluted to 500 μM and analyzed by size exclusion chromatography (SEC) toassess their apparent molecular weight and stability (i.e. theiraggregation, multimerization or degradation tendency) over time.

In a further experiment (FIG. 1C), DARPins #19 and 43 to 48 with bindingspecificity for xSA were purified to near homogeneity using theirHis-tag as described above and stored in PBS for 28 days at around 100mg/ml at −80° C., diluted to 500 μM and analyzed by size exclusionchromatography (SEC) for characterization (i.e. their aggregation,multimerization or degradation tendency). Notably, a larger column wasused compared to the first analysis series (see below).

Size Exclusion Chromatography (SEC)

Analytical SEC was carried out using a HPLC system (Agilent 1200 series)using either a Superdex 200 5/150 column (FIG. 1A and FIG. 1B) orSuperdex 200 10/300GL column (FIG. 1C) (GE Healthcare) at 20° C. TheSuperdex 200 5/150 column has a bed volume of 3.0 ml, and a void volumeof 1.08 ml (experimentally determined using blue dextran). The Superdex200 10/300GL column has a bed volume of 24 ml, and a void volume ofabout 8 ml. The measurements were performed according to standardprocedures known to the person skilled in the art. Runs were done at aflow rate of 0.2 ml/min and a maximum pressure of 15 bar (Superdex 2005/150) or 0.6 ml/min and a maximum pressure of 18 bar (Superdex 20010/300GL) in PBS. Samples of proteins were diluted in PBS to about20-500 μM, filtered (0.22 μm), and 20-100 μl of the diluted samples wereinjected on the column for separation. Elution profiles of proteinsamples were recorded by reading the absorbance at 280 nm. Aprotinin(AP) with a molecular weight of 6.5 kDa, Carbonic Anhydrase (CA) with amolecular weight of 29 kDa and Conalbumin (CO) with a molecular weightof 75 kDa were used as standard proteins to obtain a calibration curvefrom which the apparent molecular weights of the sample proteins can bedetermined.

The results are shown in FIGS. 1A to 1C. DARPins #19-22 and 27-30 showindistinguishable SEC chromatograms (i.e. indistinguishable elutionprofiles) at day 0 and day 28 of the stability study. Conclusively, allDARPins elute as monomer under the assay conditions and DARPins #19-23and 27-30 are stable for at least 1 month at 40° C. in PBS (i.e. theirelution profiles did not reveal any aggregation, multimerization ordegradation tendency).

Example 3 Thermal Stability of DARPins with Binding Specificity of xSA

Thermal stability of DARPins with specificity for xSA was analyzed witha fluorescence-based thermal stability assay (Niesen, F. H., NatureProtocols 2(9): 2212-2221, 2007). Thereby, the temperature at which aprotein (i.e. such a DARPin) unfolds is measured by an increase in thefluorescence of a dye (e.g. SYPRO orange; Invitrogen, cat. No. S6650)with affinity for hydrophobic parts of the protein, which are exposed asthe protein unfolds. The temperature at the thereby obtainedfluorescence transition midpoint (from lower fluorescence intensity tohigher fluorescence intensity) then corresponds to the midpointdenaturation temperature (Tm) of the protein analyzed.

Fluorescence-Based Thermal Stability Assay

Thermal denaturation of DARPins using SYPRO orange as a fluorescence dyewas measured using a real time PCR instrument (i.e. the C1000 thermalcycler (BioRad) in combination with a CFX96 optical system (BioRad)).DARPins were prepared at 80 μM concentration in either PBS at pH 7.4 orMES buffer at pH 5.8 containing 1×SYPRO Orange (diluted from a5'000×SYPRO Orange stock solution, Invitrogen) and 50 μl of such proteinsolutions or buffer only was added in a white 96-well PCR plate(Bio-Rad). The plates were sealed with Microseal ‘B’ Adhesive Seals(Bio-Rad) and heated in the real time PCR instrument from 20° C. to 95°C. in increments of 0.5° C. including a 25 sec hold step after eachtemperature increment, and the thermal denaturation of the DARPins wasfollowed by measurement of the relative fluorescence units of thesamples at each temperature increment. Relative fluorescence units inthe wells of the plate were measured using channel 2 of the real timePCR instruments (i.e. excitation was at 515-535 nm and detection was at560-580 nm), and the corresponding values obtained for buffer only weresubtracted. From the thereby obtained thermal denaturation transitionmidpoints, Tm values for the analyzed DARPins can be determined.

The results of the thermal denaturation of DARPins in PBS at pH7.4 orMES-buffer at pH 5.8 followed by an increase in the fluorescenceintensity of SYPRO Orange are shown in FIGS. 2A to 2D and FIGS. 3A and3B. The measured thermal denaturation transitions demonstrate that allDARPins with binding specificity for xSA analyzed have Tm values wellabove 40° C. (at both pH 7.4 and pH 5.8).

Example 4 Characterization of the DARPins with Binding for Specificityfor xSA by Surface Plasmon Resonance Analysis

DARPins with binding specificity for xSA were immobilized in a flow cellvia their His-tag to coated α-RGS-His antibody (Qiagen, cat. no. 34650),and the interaction of human, cynomolgus monkey (cyno), mouse, rat,rabbit and dog serum albumin with the immobilized DARPins were analyzed.

Surface Plasmon Resonance (SPR) Analysis

SPR was measured using a ProteOn instrument (BioRad) and measurement wasperformed according standard procedures known to the person skilled inthe art. The running buffer was PBS, pH 7.4, containing 0.01% Tween 20®.Anti-RGS-His antibody was covalently immobilized on a GLC chip (BioRad)to a level of about 2000 resonance units (RU). Immobilization of DARPinson the antibody coated chip was then performed by injecting 150 μl of 1μM DARPin solution in 300 s (flow rate=30 μl/min). The interaction withserum albumin of the different species was then measured by injecting in60 sec a volume of 100 μl running buffer (PBS containing 0.01% Tween®)containing a distinct serum albumin at a concentration of 400, 200, 100,50 nM (on-rate measurement), followed by a running buffer flow for 10 to30 minutes (flow rate=100 μl/min) (off-rate measurement). The signals(i.e. resonance unit (RU) values) of an uncoated reference cell and areference injection (i.e. injection of running buffer only) weresubtracted from the RU traces obtained after injection of the serumalbumins (double-referencing). From the SRP traces obtained from theon-rate and off-rate measurements the on- and off-rate of thecorresponding DARPin serum albumin interaction can be determined.

The results are summarized in Table 1 and Table 2. Dissociationconstants (Kd) were calculated from the estimated on- and off-ratesusing standard procedures known to the person skilled in the art andfound to be in the range from about 3 to about 300 nM. While human andcynomolgus monkey serum albumin are bound by all DARPins analyzed,rabbit, mouse, rat and dog serum albumin is only bound by a subset ofthese DARPins.

TABLE 1 Dissociation constants of DARPin serum albumin interactions Kd[nM] Kd [nM] Kd [nM] Kd [nM] Kd [nM] Kd [nM] (human) (cyno) (mouse)(rat) (rabbit) (dog) DARPin #29 15 7 n.b. n.b. 17 n.b. DARPin #20 27 110124 242 n.b. 185 DARPin #27 11 6 n.b. n.b. 19 n.b. DARPin #22 13 74  68109 n.b.  81 DARPin #28 6 3 n.b. n.b.  9 n.b. DARPin #19 14 63  56  91n.b.  77 DARPin #21 26 110 142 266 n.b. 180 DARPin #30 7 4 n.b. n.b.  8n.b. Dissociation constants for various DARPin serum albumin (fromdifferent species as indicated in each column title) interactions weremeasured by using SPR. (n.b. = no binding observable).

TABLE 2 Dissociation constants of DARPin serum albumin interactions Kd[nM] (human) DARPin #43 30 DARPin #44 39 DARPin #45 35 DARPin #46 43DARPin #47 96 DARPin #48 68 Dissociation constants for various DARPinhuman serum albumin interactions were measured by using SPR.

Example 5 Terminal Plasma Half-Life of DARPins with Binding Specificityfor xSA

The terminal plasma half-life of DARPins in mice and cynomolgus monkeys(Macaca fascicularis, also abbreviated as “cyno”) was determinedaccording to standard procedures known to the person skilled in the art(Toutain, et al., loc. cit.). A certain amount of DARPin wasintravenously injected into a mammal and the DARPin clearance from theblood plasma was followed over time by following its plasmaconcentration. The DARPin concentration initially decreases until apseudo-equilibrium is reached (alpha-phase) followed by an exponentialfurther decrease of the DARPin concentration in the plasma (beta-phase).From this beta-phase the DARPin terminal plasma half-life can then becalculated.

Determination of the DARPin Plasma Clearance in Mice

In order to assess the plasma clearance of DARPins with bindingspecificity for xSA, the test proteins were radiolabeled and injected inthe tail-vein of naïve Balb/c mice. The following DARPins were injected:DARPin#19, DARPin#21, DARPin #23, DARPin #25, DARPin #18, DARPin #32,DARPin #35, DARPin #36, DARPin #33, DARPin #34, DARPin #37, DARPin #38,DARPin #43, DARPin#44, DARPin#45, DARPin#46, DARPin#47 and DARPin#48.DARPins were radiolabeled with a ^(99m)Tc-carbonyl complex as describedpreviously (Weibel, R., et al., Nature Biotechnol. 17(9), 897-901,1999). DARPins (40 μg) were incubated with ^(99m)Tc-carbonyl (0.8-1.6 mCi) for 1 h before being diluted to 400 μl in PBS (pH 7.4). Each mousewas injected intravenously with 100 μl of the thereby obtained labeledDARPin solution (equivalent to 10 μg protein and 0.2-0.4 m Ci). Bloodsamples of the mice were collected at 1 h, 4 h, 24 h, and 48 h after theinitial injection and the radioactivity of the samples was measured. Thelevel of radioactivity measured at a certain time point is a directmeasure for the amount of DARPin still present in the blood plasma atthat time point. The % injected dose is the percentage of the totalradioactivity of the whole blood of the mouse (1.6 ml for a 18 g mouse)measured at a certain time point in relation to the total radioactivityof the injected sample corrected for the radioactive decay of ^(99m)Tc.

DARPins with binding specificity for MSA have a strongly increasedterminal plasma half-life in mice if compared to DARPin #32 having nobinding specificity for xSA (FIGS. 4A and 4B). DARPin#19, DARPin#21,DARPin#23, DARPin#33, DARPin #37, DARPin #43, DARPin#44, DARPin#45,DARPin#46, DARPin#47 and DARPin#48 had a terminal plasma half-life inmice of about 2-2.5 days.

Determination of the DARPin Plasma Clearance in Cynomolgus Monkeys

DARPin diluted in PBS were injected as a bolus injection in the cephalicvein of cynomolgus monkeys. The following DARPins were injected: DARPin#26 (0.5 mg/kg), DARPin #24 (0.5 mg/kg), DARPin #17 (0.5 mg/kg), DARPin#34 (1 mg/kg), and DARPin #32 (0.5 mg/kg). At different time pointsafter injection, plasma was generated from the blood collected from thefemoral vein of the animals. The concentration of the DARPins in theplasma samples was then determined by a sandwich ELISA using standardprotocols known to the person skilled in the art and an appropriateDARPin standard curve with known DARPin concentrations.

Plasma samples of cynomolgus monkeys were serially diluted in PBS-C (PBScontaining 0.25% casein, pH 7.4) on MaxiSorp ELISA plates that werecoated with an anti-DARPin specific rabbit monoclonal antibody. Afterextensive washing with PBS-T (PBS supplemented with 0.1% Tween 20®, pH7.4) the plates were developed with the monoclonal anti-RGS(His)4antibody labeled with horseradish peroxidase HRP (Qiagen). Binding wasthen detected by using 100 μl BM-Blue POD substrate (Roche Diagnostics).The reaction was stopped by adding 50 μl of 1 M H₂SO₄ and the absorbanceat 450 nm (and subtracting the absorbance at 620 nm) was measured. Theconcentration of the DARPin in the plasma sample was calculated byperforming a mono-exponential regression on a standard curve of theDARPin diluted in monkey serum (GraphPad Prism). The plasma terminalhalf-life of the DARPins was calculated by performing non-linearregressions (two-phase decay) on the determined concentration values upto 240 h after injection. The half-life of the second (beta) phasecorresponds to the terminal plasma half-life.

DARPin with binding specificity for xSA have an increased terminalplasma half-life in cynomolgus monkey if compared to the DARPin #32having no binding specificity for xSA (Table 3). DARPin#19, DARPin#21,DARPin #43, DARPin#44, DARPin#45, DARPin#46, DARPin#47 and DARPin#48 hada terminal plasma half-life in cynomolgus monkey of about 10 to 15 days.

TABLE 3 Estimations of terminal plasma half-life of DARPins incynomolgus monkey (cyno) t_(1/2) [h] DARPin #32 0.2 DARPin #26 129DARPin #34 111 DARPin #17 40 DARPin #24 126 DARPin #19 288 DARPin #21384 DARPin #28 144 Pharmacokinetic parameter estimates t_(1/2): terminalplasma half-life

Example 6 Higher Thermal Stability of DARPins with Improved C-TerminalCapping Modules

Thermal stability of DARPins was analyzed with a fluorescence-basedthermal stability assay as described in Example 3. Alternatively, thethermal stability of a DARPin was analyzed by CD spectrometry; i.e. bymeasurement of its heat denaturation by following its circular dichroism(CD) signal at 222 nm by techniques well known to the person skilled inthe art. The CD signal of the sample was recorded at 222 nm in a JascoJ-715 instrument (Jasco, Japan) while slowly heating the protein at aconcentration of 0.02 mM in PBS pH 7.4 from 20° C. to 95° C. using atemperature ramp of 1° C. per min. This is an effective means to followthe denaturation of DARPins as they mainly consist of alpha helices thatshow a strong change in their CD signal at 222 nm upon unfolding. Themidpoint of the observed transition of such a measured CD signal tracefor a DARPin corresponds to its Tm value.

The thermal stability of DARPin #37 (SEQ ID NO:37 with a His-tag (SEQ IDNO:15) fused to its N-terminus) was compared to the thermal stability ofDARPin #38 (SEQ ID NO:38 with a His-tag (SEQ ID NO:15) fused to itsN-terminus) using the fluorescence-based thermal stability assay. Thesetwo DARPins posses an identical amino acid sequence except for theC-terminal capping module of their repeat domains. The repeat domain ofDARPin #38, but not DARPin #37, comprises an improved C-capping moduleas described herein. The Tm values in PBS pH 7.4 determined for DARPin#37 and DARPin #38 were about 63° C. and about 73° C., respectively. TheTm values in MES buffer pH 5.8 determined for DARPin #37 and DARPin #38were about 54.5° C. and about 66° C., respectively.

The thermal stability of DARPin #39 (SEQ ID NO:39 with a His-tag (SEQ IDNO:15) fused to its N-terminus) was compared to the thermal stability ofDARPin #40 (SEQ ID NO:40 with a His-tag (SEQ ID NO:15) fused to itsN-terminus) using the fluorescence-based thermal stability assay. Thesetwo DARPins posses an identical amino acid sequence except for theC-terminal capping module of their repeat domains. The repeat domain ofDARPin #40, but not DARPin #39, comprises an improved C-capping moduleas described herein. The Tm values in MES buffer pH 5.8 determined forDARPin #39 and DARPin #40 were about 51° C. and about 55° C.,respectively.

The thermal stability of DARPin #41 (SEQ ID NO:41) was compared to thethermal stability of DARPin #42 (SEQ ID NO:42) using CD spectroscopy.These two DARPins posses an identical amino acid sequence except for theC-terminal capping module of their repeat domains. The repeat domain ofDARPin #42, but not DARPin #41, comprises an improved C-capping moduleas described herein. The Tm values in PBS pH 7.4 determined for DARPin#41 and DARPin #42 were about 59.5° C. and about 73° C., respectively.

1-16. (canceled)
 17. A method of increasing the terminal plasmahalf-life of a bioactive compound in a mammal, comprising covalentlyattaching a binding protein to said bioactive compound, wherein saidbinding protein comprises at least one ankyrin repeat domain, whereinsaid ankyrin repeat domain has binding specificity for a mammalian serumalbumin, and wherein said covalent attachment of said binding proteinincreases the terminal plasma half-life of said bioactive compound atleast 2-fold as compared to the terminal plasma half-life of saidunmodified bioactive compound.
 18. The method of claim 1, wherein saidankyrin repeat domain comprises an ankyrin repeat module having an aminoacid sequence selected from the group consisting of: (1) SEQ ID NO:49;(2) SEQ ID NO:50; (3) SEQ ID NO:51; (4) SEQ ID NO:52; (5) an amino acidsequence with up to 6 amino acids in SEQ ID NO: 49 exchanged by anyamino acid and having the ankyrin repeat sequence motif (SEQ ID NO: 53)X₁DX₂X₃X₄X₅TPLHLAAX₆X₇GHLX₈IVEVLLKX₉GADVNA 

wherein X₁ represents an amino acid residue selected from the groupconsisting of A, D, M, F, S, I, T, N, Y, and K; X₂ represents an aminoacid residue selected from the group consisting of E, K, D, F, M, N, Iand Y; X₃ represents an amino acid residue selected from the groupconsisting of W, R, N, T, H, K, A and F; X₄ represents an amino acidresidue selected from the group consisting of G and S; X₅ represents anamino acid residue selected from the group consisting of N, T and H; X₆represents an amino acid residue selected from the group consisting ofN, V and R; X₇ represents an amino acid residue selected from the groupconsisting of E, Y, N, D, H, S, A, Q, T and G; X₈ represent an aminoacid residue selected from the group consisting of E and K; X₉ representan amino acid residue selected from the group consisting of S, A, Y, Hand N; and wherein optionally up to 5 amino acids in other than inpositions denoted with X in SEQ ID NO:53 are exchanged by any aminoacid; (6) an amino acid sequence with up to 6 amino acids in SEQ ID NO:51 exchanged by any amino acid and having the ankyrin repeat sequencemotif (SEQ ID NO: 10) X₁DX₂X₃GX₄TPLHLAAX₅X₆GHLEIVEVLLKX₇GADVNA 

wherein X₁ represents an amino acid residue selected from the groupconsisting of A, D, M, F, S, I, T, N, Y and K; X₂ represents an aminoacid residue selected from the group consisting of E, K, D, F, M, N, Iand Y; X₃ represents an amino acid residue selected from the groupconsisting of W, R, N, T, H, K, A and F; X₄ represents an amino acidresidue selected from the group consisting of N, T and H; X₅ representsan amino acid residue selected from the group consisting of N, V and R;X₆ represents an amino acid residue selected from the group consistingof E, Y, N, D, H, S, A, Q, T and G; X₇ represent an amino acid residueselected from the group consisting of S, A, Y, H and N; and whereinoptionally up to 5 amino acids in other than in positions denoted with Xin SEQ ID NO:10 are exchanged by any amino acid; (7) an amino acidsequence with up to 6 amino acids in SEQ ID NO: 49 exchanged by anyamino acid and having the ankyrin repeat sequence motif (SEQ ID NO: 11)X₁DYFX₂HTPLHLAARX₃X₄HLX₅IVEVLLKX₆GADVNA 

wherein X₁ represents an amino acid residue selected from the groupconsisting of D, K and A; X₂ represents an amino acid residue selectedfrom the group consisting of D, G and S; X₃ represents an amino acidresidue selected from the group consisting of E, N, D, H, S, A, Q, T andG; X₄ represents an amino acid residue selected from the groupconsisting of G and D; X₅ represents an amino acid residue selected fromthe group consisting of E, K and G; X₆ represents an amino acid residueselected from the group consisting of H, Y, A and N; and whereinoptionally up to 5 amino acids in other than in positions denoted with Xin SEQ ID NO:11 are exchanged by any amino acid; (8) an amino acidsequence with up to 6 amino acids in SEQ ID NO: 50 exchanged by anyamino acid and having the ankyrin repeat sequence motif (SEQ ID NO: 54)X₁DFX₂GX₃TPLHLAAX₄X₅GHLEIVEVLLKX₆GADVNA 

wherein X₁ represents an amino acid residue selected from the groupconsisting of F, S, L and K; X₂ represents an amino acid residueselected from the group consisting of V and A; X₃ represents an aminoacid residue selected from the group consisting of R and K; X₄represents an amino acid residue selected from the group consisting of Sand N; X₅ represents an amino acid residue selected from the groupconsisting N, D, Q, S, A, T and E; X₆ represents an amino acid residueselected from the group consisting of A, H, Y, S and N; and whereinoptionally up to 5 amino acids in other than in positions denoted with Xin SEQ ID NO:54 are exchanged by any amino acid; (9) an amino acidsequence with up to 6 amino acids in SEQ ID NO: 50 exchanged by anyamino acid and has the ankyrin repeat sequence motif (SEQ ID NO: 12)X₁DFX₂GX₃TPLHLAAX₄DGHLEIVEVLLKX₅GADVNA 

wherein X₁ represents an amino acid residue selected from the groupconsisting of F, S, L and K; X₂ represents an amino acid residueselected from the group consisting of V and A; X₃ represents an aminoacid residue selected from the group consisting of R and K; X₄represents an amino acid residue selected from the group consisting of Sand N; X₅ represents an amino acid residue selected from the groupconsisting of A, H, Y, S and N; and wherein optionally up to 5 aminoacids in other than in positions denoted with X in SEQ ID NO:12 areexchanged by any amino acid; (10) an amino acid sequence with up to 6amino acids in SEQ ID NO: 51 exchanged by any amino acid and has theankyrin repeat sequence motif (SEQ ID NO: 13)X₁DX₂X₃GTTPLHLAAVYGHLEX₄VEVLLKX₅GADVNA 

wherein X₁ represents an amino acid residue selected from the groupconsisting of K, A, D, M, F, S, I, T, N, and Y; X₂ represents an aminoacid residue selected from the group consisting of E, K, D, F, M, N andY; X₃ represents an amino acid residue selected from the groupconsisting of R, N, T, H, K, A and F; X₄ represents an amino acidresidue selected from the group consisting of I and M; X₅ represents anamino acid residue selected from the group consisting of H, Y, K, A andN; and wherein optionally up to 5 amino acids in other than in positionsdenoted with X in SEQ ID NO:13 are exchanged by any amino acid; and (11)an amino acid sequence with up to 6 amino acids in SEQ ID NO: 52exchanged by any amino acid and has the ankyrin repeat sequence motif(SEQ ID NO: 14) X₁NETGYTPLHLADSSGHX₂EIVEVLLKX₃X₄X₅DX₆NA 

wherein X₁ represents an amino acid residue selected from the groupconsisting of Q and K; X₂ represents an amino acid residue selected fromthe group consisting of L and P; X₃ represents an amino acid residueselected from the group consisting of H, N, Y and A; X₄ represents anamino acid residue selected from the group consisting of G and S; X₅represents an amino acid residue selected from the group consisting ofA, V, T and S; X₆ represents an amino acid residue selected from thegroup consisting of V and F; and wherein optionally up to 5 amino acidsin other than in positions denoted with X in SEQ ID NO:14 are exchangedby any amino acid.
 19. The method of claim 1, wherein said ankyrinrepeat domain comprises an amino acid sequence that has at least 85%amino acid sequence identity with one ankyrin repeat domain selectedfrom the group consisting of SEQ ID NOs: 17 to 31 and 43 to 48, whereinG at position 1 and/or S at position 2 of said ankyrin repeat domain areoptionally missing.
 20. The method of claim 1, wherein said ankyrinrepeat domain comprises an ankyrin repeat module having an amino acidsequence selected from the group consisting of: (1) SEQ ID NO:49; (2)SEQ ID NO:50; (3) SEQ ID NO:51; (4) SEQ ID NO:52; (5) an amino acidsequence with up to 5 amino acids in SEQ ID NO: 49 exchanged by anyamino acid; (6) an amino acid sequence with up to 4 amino acids in SEQID NO: 50 exchanged by any amino acid; (7) an amino acid sequence withup to 3 amino acids in SEQ ID NO: 51 exchanged by any amino acid; and(8) an amino acid sequence with up to 6 amino acids in SEQ ID NO: 52exchanged by any amino acid.
 21. The method of claim 1, wherein saidankyrin repeat domain comprises an ankyrin repeat module having an aminoacid sequence with up to 5 amino acids in SEQ ID NO: 49 exchanged by anyamino acid.
 22. The method of claim 1, wherein the terminal plasmahalf-life of the bioactive compound in mice is increased to about 2 to2.5 days.
 23. The method of claim 1, wherein the terminal plasmahalf-life of the bioactive compound in cynomolgus monkeys is increasedto about 10 to 15 days.
 24. The method of claim 1, wherein the bioactivecompound is a proteinaceous bioactive compound.
 25. The method of claim8, wherein the proteinaceous bioactive compound is (1) a binding domainhaving binding specificity for a target different from mammalian serumalbumin, (2) a cytokine, (3) a growth factor, (4) an antibody or afragment thereof, or (5) a hormone.
 26. The method of claim 1, whereinthe bioactive compound is a non-proteinaceous bioactive compound. 27.The method of claim 10, wherein the non-proteinaceous bioactive compoundis a toxin, a small molecule targeting GPCRs, or an antibiotic.
 28. Themethod of claim 1, wherein said covalent attachment of said bindingprotein increases the terminal plasma half-life of the bioactivecompound at least 5-fold as compared to the terminal plasma half-life ofsaid unmodified bioactive compound.
 29. The method of claim 1, whereinsaid covalent attachment of said binding protein increases the terminalplasma half-life of the bioactive compound at least 10-fold as comparedto the terminal plasma half-life of said unmodified bioactive compound.30. The method of claim 1, wherein the terminal plasma half-life of thebinding protein in human is at least 5 days.
 31. The method of claim 1,wherein the terminal plasma half-life of the binding protein in human isat least 10 days.
 32. A method of claim 1, wherein said binding proteinhas an at least 5-fold higher terminal plasma half-life in a mammalcompared to a corresponding binding protein not binding to a mammalianserum albumin.
 33. The method of claim 1, wherein the terminal plasmahalf-life of the bioactive compound in human is increased to at least 5days.
 34. The method of claim 1, wherein the terminal plasma half-lifeof the bioactive compound in human is increased to at least 10 days. 35.The method of claim 1, wherein said ankyrin repeat domain comprises anankyrin repeat module having an amino acid sequence with up to 3 aminoacids in SEQ ID NO: 49 exchanged by any amino acid.
 36. The method ofclaim 1, wherein said ankyrin repeat domain comprises an ankyrin repeatmodule having the amino acid sequence of SEQ ID NO:
 49. 37. The methodof claim 1, wherein said ankyrin repeat domain comprises a first ankyrinrepeat module having an amino acid sequence with up to 5 amino acids inSEQ ID NO: 49 exchanged by any amino acid and a second ankyrin repeatmodule having an amino acid sequence with up to 4 amino acids in SEQ IDNO: 50 exchanged by any amino acid, wherein said first ankyrin repeatmodule is N-terminal of said second ankyrin repeat module.